J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 214 http://jad.tums.ac.ir Published Online: June 30, 2020 Original Article Evaluation of Susceptibility of Aedes caspius (Diptera: Culicidae) to Insecticides in a potent arboviral-prone Area, Southern Iran Sahar Hassandoust 1 ; *Seyed Hassan Moosa-Kazemi 1 ; *Hassan Vatandoost 1,2 ; Mohammad Mehdi Sedaghat 1 ; Kamran Akbarzadeh 1 1 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Chemical Pollutants and Pesticide, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran (Received 29 Jul 2019; accepted 20 Jun 2020) Abstract Background: Southern part of the country is a high risk for mosquito transmitted Arboviruses. This study was car- ried out to determine the base line susceptibility of the Aedini mosquitoes to the WHO-recommended insecticide. Methods: Larval collection was carried out by dipping method and adult collection occurred by suction tube from January to December 2017. The adult susceptibility test was assessed to Bendiocarb 0.1%, DDT 4%, Deltamethrin 0.05%, Lambda-cyhalothrin 0.05%, Malathion 5% and, Permethrin 0.75% at different interval times as well as at discriminative dose recommended by WHO. The larval susceptibility test was occurred using Temephos and Bacil- lus thuringiensis serotype H-14, at different concentrations. The LT50, LT90 and LC50, LC90 values were calculated for plotting the regression line using Microsoft office Excel software ver. 2007. Results: Aedes caspius was quite resistant to DDT, Malathion, Bendiocarb and showed susceptible or tolerant to other insecticides.The LT50 and LT90 values to DDT in this species were 157.896, and 301.006 minutes, respective- ly. The LC50 and LC90 values of Ae. caspius to Temephos were 0.000068, and 0.000130ppm, the figures for B. thu- ringiensis was 111.62 and 210.2ppm, respectively. Conclusion: A routine and continuous study for monitoring and evaluation of different species of Aedes to insectides is recommend at different parts of country for decision making. Keywords: Aedes caspius; Susceptibility; Iran Introduction Arthropod borne diseases are very im- portant in the world. The tribe Aedini (Fami- ly Culicidae) contains approximately one-quar- ter of the known species of mosquitoes, in- cluding vectors of deadly or debilitating dis- ease agents. This tribe contains the genus Ae- des, which is one of the three most familiar genera of mosquitoes (1). The Aedini mos- quitoes are responsible for transmission of the Barmah Forest, Batai, Babanki, Bouboui, Bun- yamwera, Chikungunya, Cache, Valley, Den- gue, Eastern Equine Encephalitis, Edge Hill, Everglades, Getah, GanGan, HighlandJ, Il- heus, James Canyon, Kedougou, La Crosse, Lebombo, Murray Valley River, Nyando, Nga- ri, Oriboca, Orungo, Pongola, Ross River, Rift Valley Fever, Semiliki Forest, Sindbis, St Louis, Encephalitis; Spondweri, Tahyna, Tensaw, Trivittatus, Uganda S, Venezuelan Equine Encephalitis, West Nile, WSLV, Wes- selbron, Wyeomyia, Yellow Fever, and Zika (1). The number of Dengue cases reported annually by WHO ranged from 0.4 to 1.3 mil- lion in the decade 1996–2005 (2). As an in- fectious disease, the number of death cases varies substantially from year to year (3). At the present, Culicidae includes; 2 sub fami- lies, 11 tribes, 113 genera and 3526 species *Corresponding authors: Dr Seyed Hassan Moosa- Kazemi, E-mail: moosakazemi@tums.ac.ir, Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com mailto:moosakazemi@tums.ac.ir mailto:hvatandoost1@yahoo.com J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 215 http://jad.tums.ac.ir Published Online: June 30, 2020 (4). The Iranian mosquitoes includes 69 spe- cies, that 7 or 11 genera depending on the classification used for aedines (5-6). Recent epidemics of mosquito-borne viral infections in countries neighboring Iran i.e. dengue, chikungunya and West Nile infections in Pa- kistan, dengue and Rift Valley fever in Sau- di Arabia, and West Nile infection in Iraq have placed this country at a serious risk for mosquito-borne diseases (7-9). Aedes caspius (Pallas) is the vector of Tahina and West Nile Viruses (7-8, 10). At the present seven Anopheles species reported as the malaria vectors in the country including: An. fluviati- liss. l, An. culicifaciess. l, An. sacharovi, An. maculipenniss. l, An. superpictus, An. stephen- si and An. dthali (11). In addition, Zaim et al. reported the An. pulcherrimusas second- ary vectors of malaria in the South East of Iran (12). Oocyte of Plasmodium found at the first time in An. multicolor, while not found in salivary glands (13). Avian malaria reported in Iran by Ghaffari (14). Spraying with residual insecticide (IRS) considered an important mosquito control measure. Twelve insecticides recommended by WHO for IRS currently, which belong to four chemical groups including one organochlorine, six pyrethroids, three organophosphates and two carbamates (15-16). DDT resistance in the adult of Ae- des aegypti, Ae. albopictus and susceptibility to Temephos, Bacillus thuringiensis and met- abolic resistance of the current species to del- tamethrin and DDT have been reported in Af- rica (17). Resistance of Ae. aegypti larvae to Temephos has been reported in Asia (18-19). In addition, larval resistance of Aedes al- bopictus to Temephos have been reported in Malaysia (20), Thailand (21).Adult suscep- tibility test on Ae. aegypti against some pay- rethroids has been reported in various research study (21-24). In spite of some reports due to resistance of An. stephensi against DDT, Dieldrin and Malathion in Iran (13, 25-31). Mechanism of resistance of An. stephensi against temephos has been reported by (32- 33). By now there are no evidence of re- sistance of Ae. vexans and Ae. caspius in Iran. Release of larvivorous fish and micro- bial agent using the Bacillus thuringiensis, and larviciding by chlorpyrifos-methyl are the main larval control measures and pyrethroid as new insecticides are being used as IRS and LLINs in Iran (34-35). In spite of more than 50 years’ malaria control programming more than 60% of the total malaria cases re- ported from Southern Iran. Malaria is one of the most important communicable diseases transmitted by anopheline mosquitoes (Dip- tera: Culicidae) to humans. In 2013, there are 97 countries and territories with ongoing ma- laria transmission, and 7 countries in the pre- vention of reintroduction phase, making a total of 104 countries and territories in which ma- laria is presently considered endemic. Based on WHO estimate, 207 million cases of ma- laria occurred globally in 2012 resulted to 627 000 deaths (2). Malaria is one of the im- portant infectious diseases in Iran with an average of about 15000 annual cases in the last decade, while total recorded cases has dropped to less than 500 locally transmitted cases in 2013. More than 80% of malaria cases in Iran are reported from three prov- inces of Sistan and Baluchistan, Hormozgan and Kerman in southern and southeastern ar- eas of the country. The most routes of ma- laria cases are immigration from Afghani- stan and Pakistan to southern and southeast- ern areas of the country (36). Over the last 20 years there has been a dramatic reduction of the malaria burden in Iran. While in 1991, nearly 100,000 cases were reported, less than 100 locally transmitted cases in 2017 (Minis- try of Health, annual reports unpublished da- ta). All observations indicate that the data re- flect the real situation and that the overwhelm- ing majority of cases, which occur, are in- cluded in the national system, although there is room for improvement in the surveillance system. The spectacular progress can be as- cribed to effective implementation of appro- J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 216 http://jad.tums.ac.ir Published Online: June 30, 2020 priate curative and preventive control inter- ventions through a strong health care infra- structure. Social and economic development allowing better housing, use of air-condition- ing etc. has also played a role. Locally trans- mitted cases are now concentrated in the south- eastern part of the country, which are affect- ed by extensive population movement across the border with Pakistan, where malaria con- trol faces serious difficulties. In 2009, Iran set time-bound elimination objectives for its ma- laria program. There has been excellent pro- gress since, but the continued risk of impor- tation of malaria cases from Pakistan poses a huge challenge, politically, socially, opera- tionally and technically, to malaria elimina- tion in Iran. The situation in the next decade will be absolute elimination or one where a few small short-lived foci emerge from time to time as a result of importation. The latest number of autochthonous cases in the whole country is 42 including 23 local malaria pa- tients, 7 relapsed cases, 12 imported from the other districts by end of July 2016 (Min- istry of Health, annual reports unpublished data). Aedes albopictus and Ae. aegypti has been recently reported in Algeria, Lebanon, Palestine, Syria, and Turkey (37). Aedes al- bopictus has been identified along the south eastern Iran (6) and Mediterranean coast of Europe for decades along with local trans- mission of DENV and chikungunya since 2007 (38). Near the Pakistan border, sero- logic evidence suggests possible DENV trans- mission in Iran (39-41), in Afghanistan (42), though local transmission has not been con- firmed to our knowledge (41). The presence of Aedes or DENV transmission in these ar- eas should not be ruled out (41). Qeshm and Kish are commercial and industrial free zones in Hormozghan Province. This area also is im- portant due to agricultural and husbandry in southern Iran in the border line of Persian Gulf and Oman sea. The study area is endemic to malaria, however in recent years, the nuisance's aedini species have been increased. There are no data about susceptibility level of Aedini vectors in Iran, so, the susceptibility level of Aedeini mosquitoes has been studied during this research. The results could provide an es- sential clue for judicious use of insecticides and will be very useful to health authorities for future planning of vector control. Material and Methods Study area The study was carried out in Hormozgan (27°11′18″N 56°16′36″E/27.1884°N 56.276 8°E), Province, southern Iran. The people en- gaged to agriculture, horticulture, livestock, fishing sailing, and hand crafts including nee- dlework, making carpet and musical instru- ments. The absolute maximum and average of temperature was reported 52 °C and 26.5 °C in Hormozghan Province, respectively. Average annual rainfall and humidity was 140.28mm and 79%, respectively. The absolute maximum and average of temperature in Isfahan was re- ported 40.6 °C and 17.1 °C. In this area aver- age annual rainfall and humidity was 63.5 mm and 22%, respectively (43) (Fig. 1). Hormozghan province with 70,697km 2 (27,296 sq. mi) square kilometers comprised of 21 counties (or districts), 69 municipalities, 13 major cities and 2,046 villages. In 2011 a little more than 1.5 million people resided in Hormozghan Province. Daregaz village (27˚ 49’GN, 56˚17’GE) with 268 households, and 926 populations, and Kovae village (27˚44 ’GN, 56˚22’GE), 38 households and 112 pop- ulations, Talsooro village (27˚46’GN, 56˚23 ’GE), 92 households and 309 populations, as fixed stations and Zakin village (27˚49’GN, 56˚16’GE), 158 households 571 population selected randomly as variable stations. Sampling methods Sampling methods such as larval collec- tion, hand catch was carried out during Jan- uary to December 2017 (44). These studies https://tools.wmflabs.org/geohack/geohack.php?pagename=Hormozgan_Province¶ms=27.1884_N_56.2768_E_type:city(1578183)_region:IR https://tools.wmflabs.org/geohack/geohack.php?pagename=Hormozgan_Province¶ms=27.1884_N_56.2768_E_type:city(1578183)_region:IR J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 217 http://jad.tums.ac.ir Published Online: June 30, 2020 were conducted once every 30 days and col- lected mosquitoes were identified by specif- ic systematic keys (5, 45). Larval collection and rearing In each fixed and variable station larvae was collected from January to December 2017. Mosquitoes larvae were picked up from the water using a dropper, pipette or fine net and inserted into the bulb. The related data such as water temperature, larval type, number and date sampling was recorded. Larvae and pu- pae in holding container filled with water were transferred to the laboratory for rearing. Mos- quito larvae feed by dry fish food. Adult mos- quitoes live quite well on bowl of sucrose 5% in bottom of the cage. The adults were kept at 28 °C, relative humidity (80%) and 14L- 10D photoperiod (46). Hand collection Aedini mosquitoes were collected from the villages between 06.30 and 09.30AM. Sam- pling was carried out in each human dwelling, cattle and goat sheds for 15min using suction tube and torch (44). The mosquitoes were trans- ferred in the cage as dimensions of 40×40cm and then sent to the laboratory. Total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel. The sucrose 5% solution was placed inside the cage. The mos- quitoes were kept in standard condition (25 ˚C, 75% RH). In Hormozghan Province, to- tally, ten species were collected including: An. stephensi, An. dthali, An. culicifacies, An. fluviatile, Cx. pipiens, Cx. quinquefasciatus, Cx. theileri, Culiseta longiareolata, Ae. cas- piuss. l and Ae. vexans. In adult collection An. stephensi was dominant species 34.76% allocated mosquitoes collected. An. dthali and An. culicifacies were followed 15%, 12.92%, respectively. Culiseta longiareolata had the lowest density with 1.09%. An. culicifacies, An. stephensi, Cx. pipiens, Cx. theileri were collected in all months. In larval collection, An. stephensi, with 1495 specimens (28.9%) was predominant followed by Cx. pipiens 753 (14.1%), An. culicifacies12.8%, Cx. quinque- fasciatus 6.3% in the same month. It should be noted that Aedes caspius larvae was col- lected in May and December. Insecticide impregnated papers Impregnated papers with DDT 4%, mal- athion0.08%, bendiocarb 0.1%, deltamethrin 0.05%, lambda-cyhalothrin 0.03%, permethrin 0.25%, and control papers were supplied by World Health Organization. Larvicides solutions. Five concentration of Temephos as (0.000015, 0.000031, 0.000062, 0.000125, 0.000250ppm) and four concentrations of Bti as (4, 36, 296, 2368ppm) were immersed in 249mL of tap water separately and larval test was applied based of WHO criteria guide- line 2016 (2). Adult susceptibility test The adult susceptibility test was carried out according WHO guideline (2). Each time 4–5 mosquito collected and insert to holding tube overall 20–25 mosquito were kept into holding tube. The susceptibility tests performed on their standard condition (22–26 °C, 60% H). The susceptibility of the wild strain of Aedini mosquitoes was assessed to the in- secticides impregnated papers. The mosqui- toes were exposed to different insecticides by different interval times and 24 hours’ re- covery period. WHO criteria for susceptibility test Based on WHO recommendations (2), the following criteria have been used for inter- pretation and classification; Mortality in the range 98–100% indicates susceptibility. A mor- tality of less than 98% is suggestive of the existence of resistance and further investiga- tion is needed. The observed mortality (cor- rected if necessary) is between 90% and 97 %, the presence of resistant genes in the vec- J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 218 http://jad.tums.ac.ir Published Online: June 30, 2020 tor population must be confirmed. The con- firmation of resistance may be obtained by performing additional bioassay tests with the same insecticide on the same population or on the progeny of any surviving mosquitoes (reared under insectary conditions) and/ or by conducting molecular assays for known resistance mechanisms. If at least two addi- tional tests consistently show mortality below 98%, then resistance is confirmed. If mortal- ity is less than 90%, confirmation of the ex- istence of resistant genes in the test popula- tion with additional bioassays may not be necessary, as long as a minimum of 100 mos- quitoes of each species was tested. However, further investigation of the mechanisms and distribution of resistance should be under- taken. When resistance is confirmed, pre-emp- tive action must be taken to manage insecti- cide resistance and to ensure that the effec- tiveness of insecticides used for malaria vec- tor control (2). Identification of mosquitoes using morpho- logical Characteristics The mosquitoes after the test were mount- ed and identified by specific systematic keys. The samples were recorded in the special forms by and the appropriate time of deaths Asso- ciated with history of collection, relative hu- midity and temperature (5, 45). Statistical analysis Results were considered reliable if the con- trol mortality was less than 5% and rejected if more than 20%. Results were corrected by Abbott's formula when mortality rates of con- trol group were between 5 to 20% (47-48). Da- ta were analyzed by probit analysis (49). Re- gression lines of the species were measured through the χ2 test. The LT50 and LT90values were calculated for plotting the regression line using Microsoft Excel software ver. 2013. Results Adult bioassay Adult bioassays using various insecticides showed that LT50 and LT90 values for DDT 4% against Ae. caspius were ranged from 157.896–301.006 minutes for the BAND strain. Bioassay test for other insecticides against is shown in Table. 1, Fig. 2. Larval bioassay Larval bioassays using Temephos showed that LC50 and LC90 for Ae. caspius ranged from 0.000068–0.000130mg/l for the BAND strain (susceptible reference strain) to 111.62–210.2 mg/L for the B. T (Table. 2, Figs. 3, 4). Mortality of Aedes caspius exposed to DDT and other insecticides has shown in Tables 1 and 2. LT50 and LT90 values of this species to DDT 4% were 157.89 and 301.006 minutes, respectively. This species was quite resistant to DDT and other insecticides ex- cept deltamethrin (Fig. 2). It is concluded that An. caspius is resistant to DDT, malathion, and bendiocarb, perme- thrin, lambdacyhalothrin whereas susceptible to deltamethrin, (Table 1). The LT50 and LT90 values of this species to DDT 4% were 157.896 and 301.006 minutes (Table 1, Fig. 2). Mortality of Aedes caspius larvae exposed to temephos and Bti has shown in (Table 2 and Figs. 3, 4). LC50 and LC90 values of this species to temephos were 0.000068 and 0.000130ppm, respectively. LC50 and LC90 values of this species to Bti were 111.62 and 210.2ppm, respectively. https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-79#Tab1 J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 219 http://jad.tums.ac.ir Published Online: June 30, 2020 Table 1. Regression line parameters of Aedes caspius adult stage exposed to some insecticides recommended by WHO in a arboviral-prone Area Southern Iran, 2017 Insecticide A B±SE LT50 95% C.I (minute) LT90 95% C.I (minute) (df) P-Value book Y= BX+A DDT4% 1.1511 0.0167±0.196 157.896 301.006 2.925 (2) P>0.05 5.99 y= 0.0167x+1.1511 Malathion 5% 1.2944 0.0081±0.190 160.229 304.435 0.289 (2) P>0.05 5.99 y= 0.0081x+1.2944 Bendiocarb 0.1% 1.6845 0.0135±0.087 42.124 80.0356 0.357 (2) P>0.05 5.99 y= 0.0135x+1.6845 Deltamethrin 0.1% 1.7745 0.0141±0.077 48.735 92.5965 0.08 (2) P>0.05 5.99 y= 0.0141x+1.7745 Lambda- cyhalothrin 0.05% 1.8494 0.0132±0.166 46.129 87.6451 11.307 (2) P>0.05 y= 0.0132x+1.8494 Permethrin 0.75% 1.5955 0.0156±0.196 29.652 56.3388 10.890 (2) P<0.05 5.99 y= 0.0156x+1.5955 Table 2. Regression line parameters of Aedes caspius larval stage exposed to Some Larvicides Recommended by WHO in arboviral-prone Area Southern Iran, 2017 Larvicide A B±SE LC50 95% C.I LC90 95% C.I P-Value book Y= BX+A Temephos 6.8275 3322.2±0.385 0.000068 0.000130 0.872 (3) P>0.05 7.81 y= 3322.2x+ 6.8275 B.thuringiensis 1.7839 0.0004±0.256 111.62 210.2 173.914 (2) P<0.05 7.81 y= 0.0004x+1.7839 Fig. 1. Map of study area, Hormozghan province, Iran J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 220 http://jad.tums.ac.ir Published Online: June 30, 2020 Fig. 2. Regression line of Aedes caspius Adult stage exposed to Some Insecticides Recommended by WHO in ar- boviral-prone Area Southern Iran, 2015 Fig. 3. Regression line of Aedes caspius larval stage exposed to Temephos Larvicide Recommended by WHO in a potent Dengue Endemic Area of Central and Southern Iran, 2015 Fig. 4. Regression line of Aedes caspius Larval stage exposed to B. thuringiensis Larvicides Recommended by WHO in arboviral-prone Area Southern Iran, 2015 J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 221 http://jad.tums.ac.ir Published Online: June 30, 2020 Discussion In our study, 4 genera and 10 species of mosquito larvae and adults were identified based on morphological characters. Culicidae species were belongs to the genus of Anoph- eles, Culex, Culisitaand Aedes. The species of Ae. caspius and Ae. vexans found by larval collection. The most predominant species was An. stephensi with 34.76% of adult and 29.36 % of larvae collection. Vatandoost et al. (2004b) (50), reported three biological forms of this species including type, intermediate and mysorensis in southern Iran. Type and in- termediate forms cited as vector in urban areas whereas, mysorensis form as vector in rural area (51). In Iran, indoor residual spraying (IRS) with DDT was carried out for malaria control during 1950–1968. In this species, re- sistance to DDT was first recognized in 1958 malathion in 1976 (13). Following the emer- gence of resistance of An. stephensi to DDT, other organophosphorus, carbamate and py- rethroid insecticides were used. The suscep- tibility level of An. stephensi to DDT and Diel- drin was studied at various parts of Iran bor- dered in Persian Gulf and Oman Sea during 1985–2016. The situation of Dengue fever and dengue hemorrhagic fever has been changed in imported to indigenous cases in Iran and probable Aedes albopictus is responsible for these endemic diseases due to unplanned ur- banization (6). In southern Iran, the climatic conditions are suitable for mosquito’s life cy- cle. The changes in temperature, humidity and wide range of water grades may have a sig- nificant effect on the population growth and also vector control programmers (52). Potent dengue vector in Iran has exophilic behavior, so, the efficacy of larvicing materials is very important to vector control programs. Temeph- os and Bti were evaluated in Lab scale against Ae. caspius larvae in the current study. In this research work, different concentrations of Bti were prepared as done by previous workers (53-54). Bacillus turingiensis is safe and ef- fective biocontrol agent used widely to con- trol of mosquitoes for the recent years (55- 58). The experiment was conducted in tape water. Abdalmagid et al. (2012) (53) checked the efficacy of Bti dunks in field water and studied the physio-chemical properties of wa- ter. They concluded that these properties have no impact on the efficacy of Bti (P> 0.05). Mulla (1990) (59) studied that it was diffi- cult to handle 1 st instar larvae because of high mortality rate during handling. Due to this rea- son we used 3 rd and 4 th instar larvae for our experiments. In the present study we found low mortality rate in case of Bti. In agree- ment with this study, Rodrigues et al. (1999) (60) reported the low mortality of Ae. agypti post treatment by Bti and 24h. Recovery pe- riods. Ramathilaga et al. (2012) (61) studied the impact of Bti against 3rd instar larvae of Ae. aegypti as was recorded in the present study against 3 rd and 4 th instar larvae of Ae. caspius. In the present study, 40% and 78% mortality was recorded for 592 and 1184ppm of Bti respectively after 24h in tape water while Ramathilaga et al. (2012) (61) record- ed (16%) mortality at the 1mg concentration of Bti for 24h treatment in tap water. Haung et al. (1993) (62) recorded 52.1, 69.5 and 78.2% mortality after 12, 24 and 48h respec- tively in 0.10ppm against Ae. aegypti larvae while 97.1, 97.1 and 97.1% mortality after 12, 24 and 48h in 0.20ppm. Gbehou et al. (2010) (63) compared the efficacy of Bti on Aedes, Culex and Anopheles species and observed 40, 80 and 100% mortality after 2, 4 and 6h against Aedes species. Many other factors such as species, genera susceptibility, feeding be- havior of larvae, instar susceptibility to bio- cides, suspended organic matter, water tem- perature, larval density, and water depth in- fluence the efficacy of Bti against mosquitoes (Boisvert 2005) (64). Some of these factors like organic, inorganic, muddy, food and floating particles decreased the efficacy of Bti due to J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 222 http://jad.tums.ac.ir Published Online: June 30, 2020 adsorption of Bti onto suspended particles fol- lowed by a slow sedimentation (65-66). In the present study, we found higher concentration of Bti is enquired for 100% mortality rate. In parallel, Ohana et al. 1987 (67), Mulla 1990 (59) reported more concentration need to con- trol of Ae. agypti larvae due to Bti a few tox- ic suspended crystals particles ingested by lar- vae. In this research study, different concen- trations of Temephos were prepared as done by previous workers (68). This larvicides is safe and effective agent used widely to con- trol of mosquitoes for the recent years. Kema- bonta and Nwankwo 2013 (68) checked the efficacy of Temephos in field water with com- parison to spinozad. They concluded that these properties have good impact on the 3 rd and 4 th Ae. agypti larvae (P> 0.05). The LC50 val- ues for wild Aedes caspius larvae were 0.000068mg/l and 0.000130mg/l, while the LC50 values for the laboratory bred and wild Aedes aegypti larvae were 7.418g/l and 8.150 g/l respectively (68). In the present study, 100% mortality was recorded at 0.000250mg/L of temephos respectively after 24h in tape wa- ter while Kemabonta and Nwankwo (2013) (68) recorded (100%) mortality at the 30g/L concentration of temephos for 24h treatment in tap water. Many other factors such as spe- cies, genera susceptibility, feeding behavior of larvae, instar susceptibility to biocides, suspended organic matter, water temperature, larval density, and water depth influence the efficacy of Bti against mosquitoes (64). Some of these Many other factors like organic, in- organic, muddy, food and floating particles decreased the efficacy of Temephos. In addi- tion, many factors effects of efficacy of Bti due to adsorption of Bti onto suspended par- ticles followed by a slow sedimentation (65- 66). In the present study, we found higher concentration of Bti will be needed for 100% mortality rate. In parallel, Ohana et al. 1987 (67), Mulla 1990 (59) were reported more concentration need to control of Ae. agypti larvae due to Bti a few toxic suspended crys- tals particles ingested by larvae. The interruption in the efficacy of Bti was found to be caused by bacterial adsorp- tion to soil particles, but the inactivation could be inverted by washing the mud away (44). Due to these reasons, the mean value of LC50 was higher against Ae. caspius larvae in com- parison to temephos. The mean LC50 values of Bti and Temephos were 111.62ppm and 0.000068ppm after 24h for tape water re- spectively. The results of the present study revealed the higher mortality post treatment by Temephos in tape water because temeph- os is considered as contact larviciding in com- parison to Bti as digestive effects and it is free of any particles due to suspended parti- cles. Based on the literature, no reports were available on the susceptibility levels of Ae. caspius. Conclusion Iran is near the Dengue endemic area, Aedes albopictus was reported for the first time in southeastern Iran in 2014. By now, IRS in human dwelling sand animal shelters, space-spraying, personal protection through distribution of LLINs and curtains (ICNs), repellents measures used to control of vec- tors in Iran. In addition, some biological and chemical agents against larval and adult stages of mosquitoes had been evaluated in the la- boratory. Results obtained from susceptibil- ity tests of Ae. caspius on some WHO recom- mended insecticides revealed that highly re- sistance to them in southern Iran. Precaution- ary measures should be taken in future vec- tor control operations. Moreover, the status of resistance in other locations in this area should be investigated. Since the country re- lies on deltamethrin for IRS operation, toler- ant populations of Aedini species implies care- ful consideration and regular monitoring of susceptibility level of mosquitoes in the future. J Arthropod-Borne Dis, June 2020, 14(2): 214–227 S Hassandoust et al.: Evaluation of Susceptibility of … 223 http://jad.tums.ac.ir Published Online: June 30, 2020 Acknowledgements The authors are grateful to Dr Majdza- deh head of National Institute of Health Re- search, Isfahan Institute of Health Research for cooperation during the study. The authors thank to Dr Holakoei-Naeini, Head of Ban- dar-Abbas Health training and Research cen- ter, for help us during this research. This study was supported the grant of SPH/TUMS/No 95-03-27-31811. The authors declare that there is no conflict of interest. References 1. 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