J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 325 http://jad.tums.ac.ir Published Online: December 31, 2020 Original Article Ecology of Malaria Vectors in an Endemic Area, Southeast of Iran *Hamideh Edalat1; Mehran Mahmoudi1; Mohammad Mehdi Sedaghat1; Seyed Hassan Moosa-Kazemi1; Sedigheh Kheirandish2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Oral and Maxillofacial Pathology, School of Dentistry, Bushehr University of Medical Sciences, Bushehr, Iran *Corresponding author: Dr Hamideh Edalat, E-mail: edalat@tums.ac.ir (Received 08 May 2018; accepted 12 Nov 2020) Abstract Background: Malaria has long been regarded as one of the most important public health issues in Iran. Although the country is now in the elimination phase, some endemic foci of malaria are still present in the southeastern areas of the country. In some endemic foci, there are no data on the malaria vectors. To fill this gap, the present study was designed to provide basic entomological data on malaria vectors in the southeastern areas of Iran. Methods: Adult and larval stages of Anopheles mosquitoes were collected by using different catch methods. Resistance of the main malaria vector in the study area to selected insecticides was evaluated using diagnostic doses advised by the World Health Organization in 2013–2014. Results: A total of 3288 larvae and 1055 adult Anopheles mosquitoes were collected, and identified as: Anopheles ste- phensi (32.1%), Anopheles culicifacies s.l. (23.4%), Anopheles dthali (23.2%), Anopheles superpictus s.l. (12.7%), and Anopheles fluviatilis s.l. (8.6%). Anopheles stephensi was the most predominant mosquito species collected indoors at the study area, with two peaks of activity in May and November. This species was found to be resistant to DDT 4%, tolerant to malathion 5% and susceptible to other tested insecticides. Conclusion: All the five malaria vectors endemic to the south of Iran were collected and identified in the study area. Our findings on the ecology and resting/feeding habitats of these malaria vectors provide information useful for plan- ning vector control program in this malarious area. Keywords: Malaria; Anopheles; Malaria vectors; Bio-ecology; Iran Introduction Malaria has long been regarded as one of the most important public health issues in Iran. The disease caused irreparable financial and fatality losses in the country, which made initiation of elimination necessary. The most important endemic foci in the country are Sis- tan and Baluchistan, Hormozgan and Kerman Provinces, which in total, account for 96% of all cases. At present, Iran is in the process of eliminating malaria, and under this condition, even low number of reported cases is very im- portant (1). Adequate understanding of the association between the behavioral characteristics of the disease vectors and their ecology is important in the planning and determination of strategies to fight against the disease. Anopheles species has been considered an important part in ma- laria transmission cycle after its role in the transmission of the disease was discovered. The ability of Anopheles mosquitoes to transmit Plasmodium infections is attributed to the phys- iology and biochemistry of their bodies, which are different according to species characteristics. Other factors such as frequency of blood feed- ing, longevity, ecological and environmental conditions are important in this respect (2). According to the last checklist of mosquitoes of Iran, there are 30 Anopheles species (3). Kerman Province has a long history of ma- Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:edalat@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 326 http://jad.tums.ac.ir Published Online: December 31, 2020 laria, and even though the disease has been well controlled in other endemic areas of the coun- try, malaria is still considered a major health problem in the province. Previous studies have identified nine Anopheles species in the prov- ince, and among them, Anopheles fluviatilis s.l., Anopheles dthali, Anopheles culicifacies s.l., Anopheles stephensi and Anopheles superpictus s.l. have been reported as vectors of malaria (4-6). Residual spraying and use of insecticide- treated bed nets have recently been implement- ed in Qaleh-Ganj County in Kerman Province. Due to the higher potential of malaria trans- mission by Anopheles vectors, it was neces- sary to carry out studies on the fauna and ecology of Anopheles species in the area and their susceptibility to some of the convention- al pesticides. The aim of this study was to col- lect information about the fauna and bio- ecology of Anopheles mosquitoes in the area, and to determine their susceptibility to some selected insecticides. Our study intended to pro- vide data that can be useful for future vector control programs in the area. Materials and Methods Study area Kerman Province is located in the southeast of the central plateau of Iran (Fig. 1). The prov- ince has a mean annual rainfall of 152.9mm, and according to the current national Counties distribution, the province is composed of 16 counties. Qaleh-Ganj is a county located in the south of the province (27.5277° N, 57.8651° E), with a population of about 70,000 people, 17% of the people live in urban and 83% in rural areas. According to the Koppen-Geiger climate classification, Qale-Ganj County is clas- sified under hot desert climate (BWh); how- ever, in recent years due to reduced rainfall, drought has gripped the area. The maximum and minimum recorded temperatures in the dis- trict are 52 and 2 °C, respectively. Monthly maximum and minimum relative humidity in the district is 70% and 35%, respectively, and rainfall in the area is between 0–125mm (Sta- tistical Yearbook of Kerman Province, 2015). Entomological survey In this study, sample collection was carried out in three villages including: Shah-Kahan (27.519126°N, 57868245°E), Marz (27.537 180°N, 57.852005°E), and Rameshk (27.521 183°N, 57.857334°E), during 2015. Study sites were chosen according to the WHO standard techniques (7). Mosquitoes were collected using the simple sampling method, before the activ- ity season of the vectors. Specimen collection was carried out over a period of 12 months using total catch, pit shelter, light trap, win- dow trap and dipping methods (7) and by us- ing night catch method during the month of May in 2015. Total catch method was used to determine species richness of each location and monthly changes in species population and diversity, and to determine the physiological status of endophagic and endophilic species. Artificial pit shelters were created for the collection of outdoor adult mosquitoes. A pit was drilled in each study location (village). Cavities with a dimension of 30×30cm were dug in the walls of the pit shelters about 0.5 meters from the floor of the shelter. These attractive cavities served as resting-sites for mosquitoes entering the pit. Mosquitoes were aspirated from the pit shelters using an aspirator, before sunrise (6– 9am) in each day of collection. Another meth- od used for mosquito sampling in our study was the light trap method. During the study, a CDC light trap was used, before sunset and until sunrise in the next day, to collect mos- quitoes in Ramsehk Village. To study the feeding and resting behaviors of adult mosquitoes, window exit traps were installed on the outside of window frames in selected locations. Collected females were iden- tified at species level and their abdominal con- dition was recorded. Night collection method using human and animal (cow) baits was con- ducted to identify the host preference of Anoph- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 327 http://jad.tums.ac.ir Published Online: December 31, 2020 eles mosquitoes in the study area. In this meth- od, mosquitoes were collected as soon as they landed on the host using torch and aspirator, from sunset to sunrise. To determine the peak of host seeking and blood feeding activity, samples collected in each hour were kept in individual cups covered by a fine net and la- beled based on the place and time of capture. After adult specimen collection, the mos- quito species were mounted on entomological pins and were identified with a morphological key (8). Female mosquitoes collected by the different methods were classified according to their abdominal condition as: gravid (G), semi- gravid (SG), unfed (U) and/or freshly blood-fed (F) (7). In the present study, anopheline mosquito larvae were collected using the dipping meth- od (7). Physical characteristics of the larval habitats were recorded during larval collection. Collected larval specimens were preserved in lacto-phenol for least 24h prior to specimen preparation for microscopic identification. The specimens were mounted on glass slides using Chloral-Gum mounting media and were cov- ered with coverslips. The glass slides were then placed in an incubator at 37 °C to dry before observing under a microscope. Identi- fication key was used to identify the mounted samples based on morphological characteris- tics (8). Susceptibility test studies Susceptibility tests were performed using insecticide-impregnated papers on 2 to 3 day old dominant Anopheles species fed with 5% sugar. Mosquitoes were exposed to insecti- cide-impregnated papers at diagnostic doses, as described by the World Health Organiza- tion pesticide scheme guidelines, for one hour, and mortality after 24-hour recovery period was recorded. The tests were carried out at a temperature between 22–26 °C and relative humidity of 60%. In the present study, DDT 4%, Malathion 5%, Propoxur 0.1% and Del- tamethrin 0.05% were used for susceptibility testing. Mortality rate of 98–100% was con- sidered as susceptible, 90–97.99% as tolerant, and < 90% as resistant (9). For each insecti- cide, four replicates of 25 2 to 3day old sugar- fed female mosquitoes were tested for suscep- tibility, whilst two replicates were used as con- trols. In this study: if control mortality was less than five percent, the results of tests were considered acceptable; if control mortality was between 5–20%, the test results were correct- ed using Abbotts' formula; and if control mor- tality was > 20%, the tests were repeated (9). Results Mosquitoes Collection A total of 3288 larvae and 1055 adult Anopheles mosquitoes were collected and iden- tified as An. stephensi (32.1%), An. culicifa- cies s.l. (23.4%), An. dthali (23.2%), An. su- perpictus s.l. (12.7%) and An. fluviatilis s.l. (8.6%). Results are described, according to the method of collection, as follows: Total catch A total of 541 Anopheles species were col- lected indoors using this method and An. ste- phensi (47.31%) was the most predominant spe- cies. The abundance of other species collected by this method is as follows: 114 (21.07%) belonged to An. culicifacies s.l., 123 (22.73%) belonged to An. dthali, 7 (1.32%) belonged to An. fluviatilis s.l., and 40 (7.57%) belonged to An. superpictus s.l. (Fig. 2). Anopheles ste- phensi was collected throughout the sampling period except in January and February. Pit shelter A total of 137 Anopheles species were col- lected from shelter pits, and An. fluviatilis s.l. was the most predominant species (35.76%). Anopheles superpictus s.l. was also identified by this method (Fig. 3). Light trap Three Anopheles species were collected by this method, and An. superpictus s.l. was the most numerically dominant. Other species col- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 328 http://jad.tums.ac.ir Published Online: December 31, 2020 lected by this method include An. culicifacies s.l. and An. stephensi (Table 1). Window trap A total of 57 female Anopheles mosquitoes representing four species were trapped by win- dow traps. Anopheles dthali (33.3%), An. cu- licifacies s.l. (31.6%), An. stephensi (26.3%), and An. superpictus s.l. (8.8%) were collected and identified using this method. Night catch using human/animal baits Table 2 shows the abundance of adult mos- quitoes collected by night catch method using human and animal baits during May 2015. Using human baits, the highest collection oc- curred in the first third of the night, and the species were identified as An. superpictus s.l. and An. fluviatilis s.l. An. culicifacies s.l. and An. fluviatilis s.l. were also collected using animal baits, with the highest collection also occurring at the first third of the night. Blood feeding peak of An. fluviatilis s.l. and An. cu- licifacies s.l. were 8–9pm and 10–11pm, re- spectively. Abdominal status of collected female mos- quitoes Abdominal condition of the female Anoph- eles mosquitoes collected by three different methods was examined. We classified the fe- males as unfed (U), blood fed (F), and gravid/ semigravid (G/SG), according to their abdominal condition (Tables 3–5). Total catch The number of gravid and semi gravid An. stephensi collected by this method was higher than the number of unfed and blood-fed An. stephensi. Unlike An. stephensi, the number of gravid and semi gravid An. culicifacies, An. fluviatilis s.l., An. superpictus s.l. and An. dthali was lower than the number of unfed and blooded ones (Table 3). Pit shelter The number of gravid and semi gravid An. culicifacies s.l. species was higher than the number of unfed and blood-fed An. culicifacies s.l. In contrast, the number of gravid and semi gravid An. stephensi, An. fluviatilis s.l., An. su- perpictus s.l. and An. dthali was lower than that of unfed and blood fed ones (Table 4). Window trap The number of gravid and semi gravid An. culicifacies s.l. and An. stephensi was higher than the number of unfed and blood-fed ones; in contrast, the number of gravid and semi grav- id An. fluviatilis s.l., An. superpictus s.l. and An. dthali was lower than the number of unfed and blood-fed species (Table 5). Larval collection Anopheles stephensi (31.59%) and An. flu- viatilis s.l. (7.29%) were respectively the most abundant and least abundant larval species collected in the study area. The results of spe- cies abundance per 10 dips have been summa- rized in Fig. 4. Characteristics of larval habi- tats in the study area have been detailed in Table 6. Susceptibility tests Mortality rate of An. stephensi against DDT 4%, malathion, propoxur, and deltamethrin was 25%, 97%, 99% and 98%, respectively. Mor- tality in the control against all insecticides test- ed were zero except for malathion and propoxur. Anopheles stephensi was quite resistant to DDT, but tolerant to Malathion and sensitive to the remaining insecticides. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 329 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 1. The number of adult mosquitoes collected by light trap in Rameshk Cllage, Qaleh-Ganj County, Kerman Province of Iran, 2015 Species J a n F e b M a r A p r M a y J u n J u l A u g S e p O c t N o v D e c T o ta l % An. stephensi 0 2 0 0 0 0 0 0 3 1 0 0 6 27.27 An. culicifacies 0 0 0 0 0 0 0 0 0 2 2 0 4 18.18 An. superpictus 0 2 3 1 0 0 0 2 0 1 3 0 12 54.55 Table 2. The abundance of mosquitoes collected on human/animal baits by night catch method, Qaleh-Ganj County, Kerman Province of Iran, May 2013 Bait Species Catch Time (Night) Total 1st third 2nd third 3rd third Human An. stephensi 7 2 1 10 An. culicifacies 5 2 2 9 An. superpictus 9 5 4 18 An. dthali 6 3 1 10 An. fluviatilis 11 6 2 19 Total 38 18 10 66 Animal An. stephensi 4 3 2 9 An. culicifacies. 15 3 4 22 An. superpictus 9 5 4 18 An. dthali 12 2 1 15 An. fluviatilis 19 6 4 29 Total 59 19 15 93 Table 3. Abdominal status of collected female mosquitoes by total catch, in the study area, Qaleh-Ganj County, Ker- man Province of Iran, 2015 T im e Species/Abdominal Status An. stephensi An. culicifacies An. superpictus. An. dthali An. fluviatilis U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l Mar 6 15 28 49 3 9 7 19 1 3 2 6 6 14 12 32 0 0 0 0 Apr 7 17 44 68 2 14 25 41 2 5 7 14 0 21 15 36 0 3 2 5 May 5 22 14 41 1 8 6 15 0 2 5 7 3 6 2 11 0 2 0 2 Jun 2 6 3 11 0 2 2 4 0 3 0 3 0 2 1 3 0 0 0 0 Jul 0 2 3 5 0 2 0 2 0 1 0 1 1 2 0 3 0 0 0 0 Aug 0 0 2 2 0 0 1 1 0 0 0 0 0 0 1 1 0 0 0 0 Sep 2 2 5 9 1 1 2 4 0 1 2 3 1 4 4 9 0 0 0 0 Oct 9 15 24 48 2 5 8 15 0 0 0 0 3 5 4 12 0 0 0 0 Nov 2 7 5 14 2 6 2 10 0 3 1 4 2 7 3 12 0 0 0 0 Dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Jan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Feb 3 3 3 9 0 2 1 3 0 2 1 3 0 3 1 4 0 0 0 0 Total 36 89 131 256 11 49 54 114 3 20 18 41 16 64 43 123 0 5 2 7 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 330 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 4. Abdominal states of collected female mosquitoes by shelter pit, in the study area, Qaleh-Ganj County, Ker- man Province of Iran, 2015 Time Species/Abdominal Status An. stephensi An. culicifacies. An. superpictus An. dthali An. fluviatilis U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l Mar 0 3 1 4 0 1 1 2 1 1 3 5 0 0 0 0 1 4 2 7 Apr 1 2 2 5 0 1 2 3 1 4 4 9 0 2 1 3 2 7 3 12 May 0 1 1 2 0 1 2 3 1 2 1 4 1 2 0 3 0 3 3 6 Jun 0 1 2 3 0 1 0 1 1 1 0 2 1 0 1 2 1 1 1 3 Jul 0 1 0 1 0 2 1 3 0 0 2 2 0 1 0 1 0 0 0 0 Aug 1 1 0 2 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 Sep 1 1 1 3 0 0 1 1 0 2 1 3 0 2 1 3 0 2 1 3 Oct 0 3 2 5 0 0 0 0 0 0 1 1 0 0 1 1 1 2 3 6 Nov 2 0 1 3 0 1 1 2 0 0 0 0 0 0 0 0 2 2 2 6 Dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 Jan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Feb 0 2 0 2 0 1 0 1 0 0 0 0 0 1 1 2 0 2 2 4 Total 5 15 10 30 0 8 9 17 4 10 12 26 2 8 5 15 7 24 18 49 Table 5. Abdominal status of collected female mosquitoes by outdoor window trap, in the study area, Qaleh-Ganj County, Kerman Province of Iran, 2015 Time Species/Abdominal Status An. stephensi An. culicifacies An. superpictus An. dthali U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l U F G + S G T o ta l Mar 0 0 2 2 0 0 1 1 0 0 0 0 0 1 1 2 Apr 0 0 1 1 0 1 2 3 0 0 1 1 0 0 2 2 May 0 0 1 1 0 0 1 1 0 0 0 0 0 0 1 1 Jun 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Jul 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 Aug 0 1 1 2 0 0 0 0 0 0 0 0 0 0 0 0 Sep 0 0 0 0 0 1 1 2 0 0 0 0 0 0 0 0 Oct 0 1 1 2 0 1 3 4 1 1 0 2 0 2 1 3 Nov 1 1 2 4 0 1 1 2 0 0 0 0 0 3 1 4 Dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Jan 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 3 Feb 0 1 2 3 1 1 2 4 0 1 1 2 0 3 1 4 Total 1 4 10 15 1 5 12 18 1 2 2 5 0 11 8 19 Table 6. Characteristics of larval habitats in the study area, Qaleh-Ganj County, Kerman Province of Iran, 2015 Categories An. stephensi An. culic- ifacies An. super- pictus An. dthali An. flu- viatilis Habitat Situation Constant 64 75 67 64 72 Temporary 36 25 33 36 28 Running Water 100 100 100 100 100 Resident Water 0 0 0 0 0 Vegetation Situation Without Vegetation 64 75 67 64 72 With Vegetation 36 25 33 36 28 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 331 http://jad.tums.ac.ir Published Online: December 31, 2020 Type Muddy 36.4 25 33 36 28 Sand 27.6 38 23 26 30 Stone or Cement 36 37 46 38 42 Water Quality Turbid 64 75 67 64 72 Fresh 36 25 33 36 28 Sunlight Situation Sunny 27.5 38 23 26 30 Semi-Shade 72.5 62 77 74 70 Shade 0 0 0 0 0 Habitat Natural 64 75 67 64 72 Artificial 36 25 33 36 28 Temperature (oC) Mean 29.5 28 26.5 31.5 22 pH Mean 7.6 7.6 7.6 7.6 7.6 Fig. 1. Study area in Kerman Province of Iran Fig. 2. The abundance of mosquitoes collected from indoors by total catch, in the study area, Qaleh-Ganj County, Kerman Province of Iran, 2015 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 332 http://jad.tums.ac.ir Published Online: December 31, 2020 Fig. 3. The abundance of mosquitoes collected by shelter pit method in the study area, Qaleh-Ganj County, Kerman Province of Iran, 2015 Fig. 4. Larval abundance per 10 dips in the study area, Qaleh-Ganj County, Kerman Province of Iran, 2015 Discussion Anopheles stephensi Among the 541 Anopheles species collect- ed indoors by the total catch method, An. ste- phensi was the most predominant species. It was highly abundant in May. In contrast to our study, An. stephensi was sampled through- out the year in another study in southeastern Iran, with the peak of activity occurring in Feb- ruary and September–October (10). In another study, the peak of An. stephensi activity was found in May and November in Bandar Abbas in southern Iran (11). However, a recent study conducted in Jask County, south of our study area, reported two peaks of activity for An. stephensi in March–April and October (12). The peak of activity of An. stephensi is direct- ly affected by weather variables especially tem- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 333 http://jad.tums.ac.ir Published Online: December 31, 2020 perature, which varies between different study areas. The density of Anopheles stephensi collect- ed in shelter pits was low. A similar result was reported in the southern Iran (10). With the night catch method, the highest number of An. stephensi mosquitoes was collected in the first third of the night between 9–10pm. In a study conducted at Khesht area in Fars Province in Iran, similar results were obtained (13). It should be mentioned that this method was only per- formed during the month of May. We recom- mend this method to be used at least in all months of the malaria transmission season in future studies in the study area. In this study, the number of gravid and semi gravid An. ste- phensi collected by shelter pit method was lower than the number of unfed and blood-fed ones, but in total catch method, the gravid and semi gravid ratio was more than 1. This shows that An. stephensi has a higher endophilic ten- dency. In a similar study conducted in Sistan and Baluchistan and Hormozgan Provinces of Iran, An. stephensi was found to be the most numerically dominant among the sampled mos- quito species. Moreover, the number of gravid and semi gravid An. stephensi collected in the study, both in shelter pits and indoors, was higher than the number of unfed and blood- fed species. The investigators indicated that An. stephensi is more endophilic compared with the other species sampled in the study areas; with G+SG/F+UF ratio lower than one both outdoors and indoors (18) Anopheles culicifacies s.l. In our study, the number of gravid and semi gravid An. culicifacies s.l. collected by total catch method was lower than unfed and blood- fed ones, but in the pit shelters and outdoor window trap methods, the gravid and semi grav- id ratio was more than 1. Moreover, the high number of unfed mosquitoes compared with gravid and blood-fed mosquitoes collected by indoor window trap method shows a high en- dophagic tendency of this species. In contrast to our findings, in a study conducted in Sistan and Baluchistan, the number of gravid and semi gravid An. culicifacies s.l. collected indoors was lower than the number of unfed and blood- fed mosquitoes. The researchers also reported the same abdominal state findings for An. cu- licifacies s.l. mosquitoes collected from pit shel- ters (14). Another study conducted in Sistan and Baluchistan indicated that this Anopheles species is more endophilic (15, 16). Consistent with our study, based on ventral aspect ratio (G+SG/U+F), another study stated that An. culicifacies s.l. prefers both indoors and out- doors as their resting places (10). Similar re- sults have also been reported in different re- gions of India (17). Although this species had two peaks of ac- tivity during March–April and October–No- vember, a study conducted in Sistan and Ba- luchistan in Iran, larvae of An. culicifacies s.l. were more abundant in April to December in rice fields with palm trees (18). In terms of breeding sites, this species was mostly col- lected from sites which have turbid water, semi-shade and have no vegetation. The mean temperature and pH of the study area were 28 oC and 7.6, respectively. An earlier study con- ducted in the south of our study area reported clear, no vegetation, sunny, and natural breed- ing sites with average temperature and pH of 25–30 oC and 7.14–8.90, respectively as cli- mate preferences of this species (19). Anopheles dthali This species was most abundant in May, June and October during the study period. A previ- ous study conducted in a relatively warmer area in Iran reported that the peak of activity of this species occurs in April and Septem- ber–October, which is one month earlier than observed in our study (20). It seems that weath- er conditions play crucial role in the period of activity of this species, making it necessary to take into consideration the weather condition of the area before planning any vector control measures. The highest abundance of this spe- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 334 http://jad.tums.ac.ir Published Online: December 31, 2020 cies, using night catch method with human and animal baits, occurred in the first third of the night between 8–9pm. We thus recommend that people be encouraged to use bed nets or avoid outdoors at these times. Larvae of An. dthali were mostly found in stagnant, turbid waters, natural breeding sites in river banks which have no vegetation, and semi-shade. In agreement with our results, this Anopheles species was sampled from breed- ing sites without vegetation (19), but unlike our findings, it was mostly collected from clear and sunny sites. Although a previous study re- ported that breeding places with temperature ranging between 13 ºC and 28 ºC and pH be- tween 6.9 and 8.0 are preferred by this species (21), the mean temperature and pH of our study sites were 31.5% and 7.6, respectively. Another study also reported that about 50% of An. dthali larvae were collected from breed- ing sites with temperature ranging between 25.1–30 oC and pH between 7.14 and 8.20 (19). Anopheles fluviatilis s.l. This Anopheles species is also considered as a secondary vector of malaria in most of its' distribution areas in Iran (22). In some stud- ies, it was captured in outdoor habitats and on animal baits (23). We found An. fluviatilis s.l. was the most predominant species (35.76%) caught by pit shelter method, confirms its ex- ophilic habit. It was also collected by night catch method using human and animal baits in May and November, mostly in the first third of the night between 9–10pm. The number of unfed and blood-fed An. fluviatilis s.l. was high- er than the number of gravid and semi gravid An. fluviatilis s.l. captured by total catch meth- od, which shows high exophilic tendency of this species. Breeding sites for this species in our study area were natural water bodies, stagnant, tur- bid waters, and semi-arid areas without vege- tation. Mean temperature and pH of the study sites were 22 oC and 7.6, respectively. This spe- cies usually breeds in fresh, slow flowing or even stagnant waters (22). Anopheles superpictus s.l. The highest collection of this species oc- curred in the first third of the night between 8 to 9pm. In our study, the number of unfed and blood-fed An. superpictus s.l. was lower than the number of gravid and semi gravid An. su- perpictus s.l. captured by total catch method, which shows high exophilic tendency of this species. Contrary to our results, a study con- ducted in south west of Iran, reported en- dophilic habit of this species in agreement with earlier studies (24). Studies on the larval habitats of An. super- pictus s.l. in Iran revealed that this species is frequently abundant in river banks, both in nat- ural breeding sites and artificial habitats creat- ed by human activities like mining pools (22). We collected this species from natural breed- ing sites, stagnant, turbid waters and semi-arid areas with no vegetation. Mean temperature and pH of the collection sites of this species were 26.5 oC and 7.6, respectively. An earlier study reported clear, sandy bed, full sunlight and nat- ural water bodies as the main breeding sites for An. superpictus s.l. (19). Insecticide resistance Our results show that Anopheles stephensi is quite resistant to DDT, tolerant to malathion and susceptible to deltamethrin and Propoxur. This result is in accordance with that of a study conducted in Chabahar (25), in which An. stephensi was found to be resistant to DDT but tolerant to malathion. Resistance of this species to DDT in Iran has been reported in the last two decades (11, 13, 26-30). Fortu- nately, in our study, this species was found to be susceptible to both deltamethrin and propoxur insecticides. Thus, these insecticides can be used in indoor residual spraying for vector control against this main endophilic malaria vector. Furthermore, it is recommended that more susceptibility tests be conducted on oth- er species with high endophilic and endophag- ic tendencies, especially An. culicifacies s.l. and An. dthali, in the area. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 325–343 H Edalat et al.: Ecology of … 335 http://jad.tums.ac.ir Published Online: December 31, 2020 Conclusion In conclusion, five malaria vectors are ac- tive in the study area. Regular entomological studies are recommended to monitor their monthly activity and susceptibility status to insecticides. The peak of host seeking activity of the indoor mosquitoes occur in the first 3rd of the night, therefore, community-based train- ing programs should be designed and imple- mented to enforce proper use of mosquito nets and personal protective measures against mos- quitoes. Country has a long history of work on ma- laria and publication of several papers on dif- ferent aspects of malaria including insecticide resistance monitoring, sibling species, molec- ular study, new record, novel methods for vec- tor control, faunestic study, use of plants for larval control, using bednets and long lasting impregnated nets, morphological studies, ma- laria epidemiology, ecology of malaria vec- tors, biodiversity, community participation, vec- tor control, repellent evaluation, anthropophilic index of malaria vectors, training is designat- ed as malaria training center by WHO. 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