J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 60 Original Article Species Composition and Seasonal Activities of Malaria Vectors in an Area at Reintroduction Prevention Stage, Khuzestan, South-Western Iran Naimatallah Maghsoodi 1, 2, Hossin Ladonni 2, *Hamid Reza Basseri 2 1Health Center of Izeh District, Jondishapour University of Medical Sciences, Ahvaz, Iran 2Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 26 Oct 2013; accepted 11 May 2014) Abstract Background: The most part of Iran become malaria-free region and fall in prevention of re-introduction stage. These regions however are struggling with imported of malaria cases where malaria vectors exist. Therefore, understanding the situation of mosquito vectors is crucial. This study was carried out to find out the present situation of malaria vectors and malaria transmission potential in a malaria-free area. Methods: The study was conducted in a malaria free area, Izeh County, Khuzestan Province during 12 months in 2011–2012. Five villages, including 2 in highlands and 3 in plain area, were selected randomly. The mosquito sam- pling methods were conducted using spray sheet and hand catch collection methods from indoor/outdoors, window trap and larvae collections. Results: In total, 3352 female Anopheles were captured, 1826 mosquito from highland and 1526 from plain areas. Five species, An. stephensi, An. fluviatilis s.l., An. dthali, An. superpictus and An. pulcherrimus were identified. The seasonal activities were started from April to March. The abdominal conditions of collected mosquitoes from in- door/outdoor places pointed to exophilic propensity of An. fluviatilis.l. s.l. and endophilic behaviour for rest of the vectors. The results of window trap also confirmed these behaviors. The larval habitats of four species were widely dispersed and included spring, margin of rivers, irrigation channels, stagnant water and rice filed. Conclusion: Understanding the present situation of malaria vectors in free-malaria area is crucial particularly where is struggling with imported cases. The results of present study can be expanded to other area of northern Khuzestan for malaria vector control planning in reintroduction prevention stage. Keywords: Malaria, Anopheles vector, Seasonal activity, Iran Introduction Malaria is very geographically specific and remains an important cause of mortality and morbidity in many parts of the world. In 2012, there were 99 countries and territories which highly struggling malaria transmission and 5 countries in the prevention of reintro- duction phase (WHO 2013). Although this disease has been eradicated from many re- gions, there is more possible to reintroduce to an area particularly where Anopheles vec- tors are present circumstances are suitable for parasite transmission (Roll Back Malaria 2013). Therefore, to prevent malaria reintroduction is a big challenge (Hemami et al. 2013). Malaria was native in the most parts of Iran but after six decade effort, the disease has been limited to south-eastern part of the country (Raeisi et al. 2013). Anti-malaria op- eration was started in Iran in 1945 and which cause malaria infection rate considerably de- creased during 1948–1956 in the most endem- ic areas. Later malaria eradication programme started and since 1980 almost interrupted ma- laria transmission in the north and middle parts of the country. Then the programme shifted to malaria control programme which has been *Corresponding author: Dr Hamid Reza Basseri, E- mail: basserih@tums.ac.ir http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 61 continuing up to present time (Azizi and Bahadori 2013). Seven Anopheles including An. maculipennis s.l., An. sacharovi, An. superpictus, An. fluviatilis s.l., An. stephensi, An. culicifacies and An. dthali are considered to be main malaria vectors in Iran (Zahar 1974, Hanafi-Bojd et al. 2011). In addition, An. pulcherrimus has been reported as sus- pected potential vector of malaria in the district of Ghassreghand, Baluchistan, Iran (Zaim et al. 1993). More recently four Anoph- eles species, An. stephenis, An. dthali, An. pulcherrimus and An. superpictus have been reported from Mahshar County located in south of Khuzestan Province, Iran (Farhadinejad et al. 2013). At present, Iran fall to pre-elimination of malaria but the main challenges to sustaining elimination are addressing the potential re- introduction of cases, either via long border with malaria-endemic countries at east or from migrant populations scatter to whole country (Raeisi et al. 2013). However, malaria trans- mission is highly depend on community and Anopheles vectors behavior particularly in southern Iran where is threaded by imported cases (Shahandeh et al. 2010). Khuzestan Province recently became free from malaria transmission but the area is threatened by imported malaria cases (Akbari et al. 2013). In this circumstance, vector con- trol plays a main role to prevent malaria out- break. Therefore, understanding the vectors' situation in advance is so essential for ham- pering of malaria occurrence. Izeh County is located at north of Khuzestan known as a malaria eliminated zone. Due to travel of job seeker labors or immigrants into the county, reintroduction of malaria particularly cases that lead to re-establishment of local trans- mission is the most important issue (personal communication with Izeh district Health Cen- tre). Izeh has suitable environments for mos- quito development but for more than five years no any malaria transmission has been reported, therefore, vector control programmes have been limited to larviciding operation and environmental management (County Health Centre of Izeh, personal communication). The present study illustrates the current situation of malaria vectors and their activi- ties in the Izeh County with a scope of role of the vector in reintroduction of malaria. The results of this study can be used for sim- ilar area which has imported malaria cases and potent vectors. Materials and Methods Study area Izeh County is located at North of Khuzestan Province. The county is situated in slope of Zagrus Mountain chain at north and Khuzestan Plain in south (Fig. 1). It is ge- ographically located on latitude 31°34′8″N, and longitude 49°34′0″E. Generally, the area comprises mountainous, hilly regions in the north, with plains regions in the south and west. The region has a mild climate and tem- perature reaches to 40 °C at maximum in sum- mer and minimum near 10 °C in December and January. The water sources are perma- nent and temporary rivers, wells springs, ponds and pools. The annual rainfall ranged from 100 to 120 mm. The rainfall occasion- ally received during winter. Izeh has Primary Health Care (PHC) since 1988 and all rural and urban areas have PHC services. By integrating malaria control ac- tivities into health system, the health work- ers involved in malaria cases finding as well as vector control programme. Mosquito collection Mosquito collections were standardized as fully described by WHO (WHO 2003). During the 12 months, mosquitoes were col- lected from 3 villages located in mountain- ous lands and 2 villages in plain lands. Col- lections were carried out from eight indoor and outdoor shelters in each village as follow. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 62 Indoor collection The mosquitoes were collected from hu- man dwellings and sheep or cattle sheds by pyrethrum space spray method as fully de- scribed by Service and WHO (Service 1976, WHO 2003). Briefly, before spray, all the eves, windows, doors, and other exit points in each indoor shelter were closed and then white cloth sheets were spread on the floor. Pyrethrum extract (0.2% in kerosene) was sprayed in the entire space of the room and the room was closed for 15 minutes. After 15 minutes, all the knocked-down insects lying on the cloth sheet were collected care- fully with the forceps and placed in petri dishes lined with moist filter paper and brought to the laboratory for further studies. The time of application in each village was early in the morning and during sunrise be- tween 0530 and 0730 h. Outdoor collection Natural shelters inside and around the in- dicator villages were periodically searched during early morning and mosquitoes were collected using sucking tube and torch light. The mosquitoes were then captured using sucking tube and touch. All mosquitoes were identified based on species taxonomic keys of Shahgudian (Shahgudian 1960). All details of the shelters including kind of habits, temperature, humidity, and date and time of collection were recorded on forms. In addition, collected female mosqui- toes were graded to abdominal conditions in each sampling technique as described by (WHO 2003). Generally, endo/exophilic be- havior of each mosquito species was catego- rized based on abdominal appearance of the collected mosquitoes as follows. The gravid (G) and/or semi-gravid (SG) appearance of the female abdomen demonstrate as resting stages of female mosquitoes, and the females with unfed (U) and freshly fed (F) guts are indicative of the seeking stages (seeking for blood meal or resting places). Therefore, ratio of the resting stages classified as tendency to rest in indoor or outdoor places (WHO 2003). Window trap collection Both entry and exit window traps (Length x width x height= 30× 30× 30 cm) were uti- lized to find moving behavior of mosquitoes in to the indoor shelters. Two entry and two exit traps were fixed on the windows of se- lected rooms in each villages from sun set until sun rise. All mosquitoes were collected alive with sucking tube and transferred to laboratory for identification. Larval sampling and processing Larval surveillance was conducted twice a month over a 12 month period in 2011. The larvae collection was carried using standard mosquito dipper from all potent breeding places out around the selected villages with a radius of about one kilometer. All collected larvae were counted and III-IV instars larvae trans- ferred to lacto-phenol. The larvae were then identified 24 hours after conservation using morphological taxonomic key (Shahgudian 1960). Results In total, 3352 female Anopheles spp. were captured including 1826 mosquitoes from mountainous and 1526 from plain areas. The mosquito species were An. stephensi (ntotal= 837, nindoor= 701, noutdoor= 21, Window traps= 115), An. superpictus (ntotal= 1100, nindoor= 871, noutdoor= 48, Widow traps= 181), An. fluviatilis s.l. (ntotal= 326, nindoor= 129, noutdoor= 126, window traps= 71), An. dthali (ntotal= 1088, nindoor= 993, noutdoor= 47, Window traps= 137) and An. pulcherrimus (nindoor= 1). As illustrated in Fig. 1, the seasonal pop- ulation dynamics of the mosquitoes in in- door places were varied among four species either sampled from mountainous or plain http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 63 areas. Totally, An. stephensi, An. superpictus and An. dthali population trend in mountain- ous followed the same pattern in plain areas (Fig. 1A, B, C) but An. fluviatilis s.l. had low indoor abundance in the plain areas com- paring with mountainous areas (Fig. 1D). The seasonal activities of four species were started in April and the highest population of An. superpictus s.l. and An. dthali was ob- served in June while the maximum popula- tion of An. stephensi and An. fluviatilis s.l. occurred in October. An. superpictus exhib- ited more tendencies to be in indoor shelters (Fig. 1B) followed by An. stephensi and An. dthali populations (Fig. 1A). Generally, the population density of adults declined to min- imum in winter. Abundance of female mosquitoes collect- ed from animal and human shelters was var- ied in either highland or low land areas (Ta- ble 1). Anopheles stephensi showed slightly more tendency to human shelters rather than animals while An. superpictus, An. dthali and An. fluviatilis s.l. were captured more in animal shelters comparing with human plac- es. Totally, the pattern of using human/ animal shelters as resting places by each species in highland areas was same as lowlands (Table 1). Four species also showed propensities for outdoor shelters (Fig. 2). The population dy- namics of mosquitoes in outdoor shelters dis- placed similar pattern to indoor places. The mosquitos’ activities started from April to De- cember or January. Anopheles fluviatilis s.l. showed more propensities for resting in out- door shelters either in plain or mountain ar- eas (Fig. 2D). In addition, female mosquitoes were graded according to abdominal conditions collected indoors and outdoors. The gravid (G) and/or semi-gravid (SG) appearance of the abdo- men demonstrate as resting stages, while the female mosquitoes with unfed guts (U) and/ or freshly fed (F) are indicative of the seek- ing stages. The ratio of resting stages to seeking stages for An. stephensi showed that this species had a greater tendency to rest inside (G, SG/E, F= 0.75) rather than out- doors (G, SG/E, F= 0.50). Anopheles fluviatilis s.l. had a low proportion for endophilic be- havior (G, SG/E, F= 0.17) and its preference for resting outside (G, SG/E, F= 0.56) was five times more than An. stephensi and near- ly two times more than An. superpictus (Ta- ble 2). Anopheles superpictus showed slight- ly more exophilic behavior (G, SG/E, F= 0.71) than endophililc (G, SG/E, F= 0.73). Though the majority of An. dthali was col- lected in indoor shelters, resting tendency to indoor (G, SG/E, F= 0.88) was more than outdoors (G, SG/E, F= 0.51). The number of mosquitoes caught by window traps is showed in Table 3. Gener- ally, indoor tendency exhibited by four Anoph- eles species either in higher in lowland or highland. Entry behavior of An. stephensi, An. superpictus, An. dthali and An. fluviatilis s.l. was observed during whole activity sea- sons (Fig. 3). The dynamic of entry move- ment of four Anopheles species was alike to population dynamics of them sampled from indoors. Totally, 931 larvae were collected from plain (418 larvae) and mountainous (513 larvae) areas. The collected larvae species in plain area were An. stephensi (117 larvae), An. superpictus (170 larvae), An. dthali (80 larvae) and An. fluviatilis s.l. (51 larvae) and in mountainous area An. stephensi (160 lar- vae), An. superpictus (105 larvae), An. dthali (222 larvae) and An. fluviatilis s.l. (26 larvae). The larvae were generally found in spring, streams, margin of rivers, irrigation channels, stagnant water such as waste water, drain- age, borrow pits as well as rice fields. The ratio of Anopheles species in each type of breeding places was highly varied. In addi- tion high different between mountainous and plain areas was observed with respect to using different breeding places (Fig. 4, 5). Anopheles stephensi larvae were found mostly in stagnant waters or waters with low flow http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 64 rate such as rice farm. Comparing to larvae combination in each type of breeding place, An. stephensi larvae ratio in the lowland area (plain) was mostly rice farm (Fig. 3C) while in the highland, it found mostly in springs. Anopheles dthali larvae collected in various breeding places with relatively high ratio comparing with other Anopheles mosquitoes (Fig. 4, 5) except in rivers margin of highland region (Fig. 4A). In contrary, high abundance of An. superpictus was collected in river margins particularly in highland (Fig. A). Anopheles fluviatilis s.l. was found at low ratio in stagnant waters and spring of high- lands while more ratio of An. fluviatilis.l. s.l. larvae (19%) was collected in stagnant wa- ters of in plain areas (Fig. 3B, Fig. 4B, C). Table 1. Number of female mosquito captured from human and animal places in two different areas, Izeh County, Southwest of Iran, 2011–2012 Species Mountainous areas Total Plain areas Total Animal shelters (%) Human shelters (%) Animal shelters (%) Human shelters (%) An. stephensi 230 (48.1) 248 (51.8) 478 103 (46.2) 120 (53.8) 223 An. dthali 302 (63.8) 17 (35.2) 473 299 (57.5) 221 (42.5) 520 An. superpictus 249 (50.1) 244 (48.9) 498 201 (53.8) 172 (46.1) 373 An. fluviatilis s.l. 46 (38.3) 74 (61.6) 120 9 (100) 0 (0.0) 9 An. pulcherrimus 0 0 0 1 (0.002) 0 1 (0.0008) Table 2. Abdominal condition of female mosquitoes based on collecting sites in Izeh County, southwest of Iran, 2011–2012 Species Indoor collections G,SG/F, E (indoors) Outdoor collections G,SG/F, E (outdoors) Outdoors/ IndoorsF, E G,SG F, E G, SG An. stephensi 399 (57.1%) 302 (42.9%) 0.75 14 (72.2%) 7 (27.8%) 0.50 0.66 An. superpictus 504 (57.8%) 367 (42.2%) 0.73 28 (64.7%) 20 (35.3%) 0.71 0.97 An. fluviatilis s.l. 110 (85.3%) 19 (14.7%) 0.17 81 (70.0%) 45 (30.0%) 0.55 3.27 An. dthali 528 (53.21%) 465 (46.8%) 0.88 31 (68.1%) 16 (31.9%) 0.52 0.59 F: Fresh Fed female mosquito E: Empty or Unfed female mosquito G: Gravid female mosquito SG: Semi-Gravid female mosquito Table 3. Frequency of female mosquito captured from exit andentry window trap in two different areas, Izeh County, southwest of Iran, 2011–2012 Species Mountainous areas Total Plain areas Total Exit (%) Entry (%) Exit (%) Entry (%) An. stephensi 11 (34.3) 21 (65.5) 32 32 (38.5) 51 (61.4) 83 An. dthali 17 (36.1) 30 (63.8) 47 46 (51.1) 44 (48.8) 90 An. superpictus 40 (61.5) 25 (38.4) 65 62 (53.4) 54 (46.5) 116 An. fluviatilis s.l. 23 (62.1) 14 (37.8) 37 26 (76.4) 8 (23.5) 34 http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 65 Fig. 1. Seasonal prevalence of female mosquitoes: Anopheles stephensi (A), An. dthali (B), An. superpictus (C) and An. fluviatilis s.l. (D) collected from indoor places in both mountainous and plain areas of Izeh County (2011–2012). The population trends of mosquitoes were similar in both distinguishable areas Fig. 2. Seasonal density of female mosquitoes: An. stephensi (A), An. dthali (B), An. superpictus (C) and An. fluviatilis s.l. (D) collected from outdoor places in both mountainous and plain areas of Izeh County (2011–2012) http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 66 Fig. 3. Mean number of female mosquitoes, Anopheles stephensi (A), An. dthali(B), An. superpictus (C) and An. fluviatilis s.l. (D) collected by exit and entry window traps in Izeh County (2011– 2012). The population trends of mosquitoes were similar in both distinguishable areas Fig. 4. Larvae ratio of four Anopheles species, col- lected from different habitants in plain areas A: River side B: Stagnant water C: Rice farm D: Spring Fig. 5. Larvae ratio of four Anopheles species col- lected from different habitants in mountainous area. A: River side B: Stagnant water C: Spring Discussion Indeed, mosquito collections were carried out on probably occurrence of malaria out- breaks in an eliminated zone such as north of Khuzestan Province not based on a more com- prehensive seasonal or temporal collection pro- tocol may have resulted in a lower mosquito vectors activities overall. However, our re- sults indicate that the activities and behavior http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 67 of four Anopheles vectors in Izeh County in- dicates that the area may have potent for ma- laria transmission depend on mosquito vec- tors contact with any malaria cases. Totally, the population trend of three mos- quito species in mountainous area followed the same pattern in plain (Fig. 1A, B, C). In contrary, An. fluviatilis s.l. had low indoor abundance in the plain area rather than moun- tainous. These results may indicate that the environmental condition in both areas is sim- ilar for endophilic species. In the present study, An. superpictus and An. dthali were the most abundant and prev- alent species showing preference to be indoor places while An. fluviatilis s.l. at low preva- lent noticeably to outdoor places. The abun- dant of An. stephensi was also high in both indoor and outdoor shelters with more in- tendancy to indoors in Izeh areas. Anopheles fluviatilis s.l. was collected at very low fre- quency in indoor places particularly in plain areas indicating that this mosquito is more exophlic and this has been confirmed by the results of collection using window trap meth- ods. Similar behavior of An. fluviatilis s.l. was declared by several authors (Viswanathan et al. 1945, Eshghi et al. 1976, Nanda et al. 1996, Sahu et al. 2009, Sahu et al. 2011). It was illustrated that An. fluviatilis s.l. as a wild mosquito had relatively high tendency to human blood (Manouchehri et al. 1976a, Basseri et al. 2010). Totally, it seems host seeking behavior of four Anopheles adults may slightly different when they exhibited varied movement and resting behaviour in Izeh County. We also found that larval habitats of An. fluviatilis s.l. may be small, widely dispersed, and transient, and this may be reason we could not find the larvae at high abundance in Izeh County. Overall, control of An. fluviatilis s.l. is big challenge due to the be- havior of this mosquito vector in Izeh Coun- ty. However, this mosquito considered as one of the most important malaria vector for sta- ble malaria in Middle East and south of Asia countries (Eshghi et al. 1976, Manouchehri et al. 1976a, Gunasekaran 1994, Dev et al. 2003). Anopheles stephensi was recognized as a domestic mosquito (Sharma 1995, Kar et al. 1996, Chakraborty et al. 1998). It was respon- sible for malaria epidemic in some part of southern Iran (Manouchehri et al. 1976b). In addition, this species exhibits a strong pref- erence for human blood in south and south eastern Iran (Basseri et al. 2005, 2010). In the present study, An. stephensi showed endophilic behavior as well as using natural shelters for rest. Therefore, at outbreak situation of ma- laria, using indoor residual spraying may not be effective method for control of this vector, particularly this anopheles has become re- sistant to several insecticides in Iran (Enayati et al. 2003, Vatandoost et al. 2006). Anopheles dthali was identified as sec- ondary malaria vector in Iran (Manouchehri et al. 1972). As in previous study stated this mosquito use human dwelling as well as an- imal shelter in south of Iran (Manouchehri and Rohani 1975), we also found An. dthali in the same places at relatively high abun- dance. Anopheles dthali is still susceptible to all insecticide which used for indoor residual spraying (Vatandoost et al. 2007). However, it was stated that An. dthali can be incrimi- nated for transmission malaria at high den- sity where we comparatively found in Izeh County. In the present study, Anopheles superpictus was found relatively at high density in both mountainous and plain areas of Izeh County. This mosquito was also captured by window traps more than other mosquito vectors. Anopheles superpictus is one of the most widespread malaria vectors in Iran (Hanafi- Bojd et al. 2011). Overall, the results ob- tained from window traps collection method (Table 3) and density of the female mosquito per room (Fig. 1) as well as abdominal con- dition ratio (Table 2) together indicating that this species has more propensity to indoor http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 60–70 N Maghsoodi et al.: Species Composition … 68 places than outdoors. Therefore, in any ma- laria outbreak situation, the behavior of An. superpictus should be considered as a potent vector in north of Khuzestan Province. This species had been responsible for maintaining malaria transmission due to its refractory be- havior in southern slopes of the Zagros chain (Zahar AR 1990). The study area is very fertile with differ- ent water source for breeding of Anopheles larvae. The agricultural activities start from beginning of spring and provide many jobs for immigrant labors. Furthermore, the envi- ronment of Izeh provides suitable condition for activity of four malaria vectors. Accord- ing to report of Izeh Health Centre, the area is struggling with imported malaria cases an- nually. Therefore, occurrence of malaria trans- mission should be expected. Conclusion Although malaria has been eliminated from north of Khuzestan, but due to population movement form malaria endemic area, un- derstanding the present situation of malaria vector is essential. The results of present study can be expanded to other area of northern Khuzestan for malaria vector control plan- ning in reintroduction prevention stage. Acknowledgements We thank the personnel of the Izeh Health Centers for kind assistances. The authors would like to thank Dr Ahmad Raeisi, the Head of Malaria Control Office, Ministry of Public Health, for his great technical sup- ports. 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