J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 398 http://jad.tums.ac.ir Published Online: December 25, 2018 Original Article Seasonal Activity of Adult Mosquitoes (Diptera: Culicidae) in a Focus of Dirofilariasis and West Nile Infection in Northern Iran *Shahyad Azari-Hamidian 1, 2, Behzad Norouzi 1, Ayoob Noorallahi 3, Ahmad Ali Hanafi- Bojd 4 1Research Center of Health and Environment, Guilan University of Medical Sciences, Rasht, Iran 2School of Health, Guilan University of Medical Sciences, Rasht, Iran 3Department of Disease Control and Prevention, Health Vice-Chancellorship, Guilan University of Medical Sciences, Rasht, Iran 4Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 9 Jun 2018; accepted 18 Nov 2018) Abstract Background: Mosquito-borne arboviruses such as West Nile, dengue, Rift Valley fever, and Sindbis viruses and the nematode Dirofilaria are reported in Iran, but there is little information on the seasonal activity of their vectors in the country. We aimed to determine the seasonal activity of adult mosquitoes (Diptera: Culicidae) in a focus of diro- filariasis and West Nile infection in Guilan Province, northern Iran. Methods: Collections were carried out using light traps in seven counties at least two times from random sites and every two weeks from a fixed site (Pareh Village, Rudbar County) during Aug–Dec 2015 and Apr–Oct 2016. Results: Overall, 16327 adult mosquitoes comprising 18 species representing seven genera were identified. The most prevalent species were Cx. theileri (23.59%), Cx. tritaeniorhynchus (20.75%), Cx. pipiens (19.37%), Ae. vexans (18.18%), An. pseudopictus (10.92%) and An. maculipennis s.l. (5.48%). Aedes pulcritarsis and Cx. perexiguus were found for the first time in Guilan Province. The active season of adult mosquitoes extended from early May to early Oct in the fixed site. There was no significant regression between the abundance of adult mosquitoes and the meteor- ological data during active season in the fixed site (P> 0.05, R2= 0.31). Conclusion: Though no significant regression between the abundance of mosquitoes and the meteorological data was observed during active season, temperature and rice fields had a great influence in starting and ending active season in the region. Keywords: Dirofilaria, Flavivirus, Flaviviridae, Vectors, Iran Introduction West Nile virus (WNV) (Flaviviridae: Fla- vivirus) and its subtype Kunjin is distributed in Eurasia, Africa, North and Central America and Australia. Mosquitoes (Diptera: Culicidae), es- pecially ornithophilic species, are the principal vectors of the virus and some virus isolations have been reported from soft and hard ticks. Wild birds, especially wetland species, are the principal vertebrate hosts, the virus has also been isolated from mammals and frogs (1, 2). WNV infection is recorded from horses in at least 26 provinces (out of total 31) in Iran (3–5), humans (6–12) and birds (13). Guilan Province in the Caspian Sea littoral of north- ern Iran, with vast wetlands, is one of the foci of WNV where infections are found in humans (1.4–10%) (4, 7–8), horses (2.2–25%) (3–4) and birds (especially the common coot, the main reservoir) (62.7%) (13). Recently, the vi- rus was found in Aedes (Ochlerotatus) caspius (Pallas) s.l. [Ochlerotatus caspius s.l.] in West Azerbaijan Province, northwestern Iran, and *Corresponding author: Dr Shahyad Azari- Hamidian, E-mail: azari@gums.ac.ir J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 399 http://jad.tums.ac.ir Published Online: December 25, 2018 in Cx. pipiens Linnaeus in Guilan Province, northern Iran, respectively (14, 15). Dirofilariasis is a disease caused by dif- ferent species of the nematode genus Dirofi- laria (Spirurida: Onchocercidae), especially D. immitis (canine or dog heartworm) and D. re- pens, transmitted by mosquitoes. The disease is cosmopolitan. The reservoirs of the nema- todes are many different mammals, especial- ly canids. Previously, human dirofilariasis (HD) was considered a rare disease, but at the pre- sent time, HD is classified as an emerging dis- ease in some areas because the number of re- ported cases was dramatically increased (16). Dirofilariasis is found in humans, dogs, wolves, jackals, foxes and cats in at least 15 provinces of Iran (17–22). Guilan Province is one of the foci of dirofilariasis, where D. re- pens infection is found in humans (17, 23) and D. immitis found in 4.4% (24) to 51.4% of local dogs (25, 26). Culex theileri Theobald is a known vector of D. immitis in northwest- ern Iran (27). The last checklist of Iranian mosquitoes comprises 64 species and seven genera (28, 29). Subsequently, Anopheles superpictus Grassi includes two species in Iran based on the In- ternal Transcribed Spacer 2 (ITS2) sequences of rDNA (30), later listed as species A and B (31). A new species of the Anopheles hyrca- nus group (An. hyrcanus spIR) was recognized from southwestern Iran, also based on ITS2 sequences (32). More recently, the occurrence of Aedes (Stegomyia) albopictus (Skuse) [Steg- omyia albopicta] and Ae. (Stg.) unilineatus (Theobald) [Stegomyia unilineata] were report- ed in southeastern Iran and Orthopodomyia pulcripalpis (Rondani) in northern Iran, respec- tively (33–35). Overall, 30 species of mosqui- toes representing seven genera were listed in Guilan Province (36). A large amount of available data on mos- quitoes in Iran is based on collections and ecol- ogy of larvae (27, 36–40 and many other ref- erences cited by aforementioned articles). Dif- ferent methods of collecting adult mosquitoes, such as using light traps, aspirators, pit shelters and total catch (Pyrethrum space spray), have been used mostly in relation to anopheline vec- tors of malarial protozoa (41–47). There are a few published documents in the country that deal with adult sampling, especially using light traps, which include culicines (27, 48–50), but there are no studies of seasonal activity. That is why there is very little information about the seasonal activity of culicine adults in Iran. This study was carried out by means of light traps to study the seasonal activity of mos- quitoes, especially probable and proven vectors of WNV and Dirofilaria, in Guilan Province, northern Iran. Materials and Methods Study area Guilan Province locates in the Caspian Sea littoral of northern Iran, between the Caspian Sea and the Alborz Mountain range. It has coastal, plain, foothill, and mountainous are- as with an area of approximately 14,700 square kilometers. The province is bordered by Ma- zandaran Province in the east, Ardabil Prov- ince in the west and Zanjan and Qazvin prov- inces in the south. It is also bordered by the Republic of Azerbaijan in the north as well as Russia across the Caspian Sea (Fig. 1). The province has a temperate climate and relatively warm-humid summer. It is located between 36o33'–38o27' N latitude and 48o32'–50o36' E longitude and formally includes 16 counties. Most areas of Guilan Province with about 1000–2000mm of rainfall annually, have the greatest amount of rainfall in Iran, and the main agricultural crop is rice. The province has vast deciduous forests of Hyrcania, temperate cli- mate, vast wetlands and rice fields, which pro- vide abundant habitats for mosquitoes. Specimen and data collection In seven counties (including different topo- graphical areas of the province) adult collec- tions were carried out at least two times from J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 400 http://jad.tums.ac.ir Published Online: December 25, 2018 random (variable) sites during Aug–Dec 2015 and Apr–Oct 2016 (Fig. 1, Table 1). Sampling was also carried out at a fixed site (Pareh Vil- lage of Rudbar County, 36o 50.800' N, 49o 32.650' E, altitude 487m) every two weeks from Apr to Oct 2016. Rudbar County in south- ern Guilan has about 200–500mm annual rain- fall and showed mountainous and less humid temperate climate similar Mediterranean Re- gion. Pareh Village is in a foothill area and close to natural Hyrcanian forest and manmade woodland that includes trees such as olive, wal- nut, fig, Persian ironwood (Parrotia persica) and Caucasian elm or Caucasian zelkova (Zelko- va carpinifolia). The main livelihood of the people in the village is husbandry and the main domestic animals are cattle and sheep. Dogs, horses, donkeys and fowls are also common an- imals in the region. The meteorological data of Pareh Village during 2016 is shown in Table 2. Two CDC light traps were used in each var- iable and fixed site. The light traps were sus- pended from the ceiling in animal shelters from sunset to sunrise, i.e. from 1800 to 0600 hrs. The electricity of traps was provided by 6-volt rechargeable batteries. Moreover, ad hoc col- lections were carried out using manual aspi- rators (hand catch) in the fixed and random sites. The specimens were identified using the mor- phological-based keys (29). The abbreviations of mosquito genera and subgenera follow Reinert (51). The specimens are deposited at the Mu- seum of Medical and Veterinary Entomology, School of Health, Guilan University of Med- ical Sciences, Rasht, Iran. Determining species dominance structure The dominance structure of a species is expressed as the percentage of specimens of the species in the whole sample. The follow- ing five percentage representation categories (52, 53) were used: Eudominat (ED) species (> 30%), dominant (D) (10–30%), subdomi- nant (SD) (5–10%), recedent (R) (1–5%) and subrecedent (SR) (< 1%). Mapping collected mosquitoes and statistical analysis ArcGIS 10.3 was used to create a geo-da- tabase of mosquitoes and to map the collec- tion sites and the distributions of the most med- ically important species. The statistical analy- sis of mosquito abundance and meteorological data was carried out using the linear regression test of SPSS software (ver. 16 for Windows, SPSS Inc., Chicago, IL). Results Mosquito fauna Overall, 16327 adult mosquitoes were col- lected during 29 surveys (Aug–Dec 2015 and Apr–Oct 2016): 15959 (97.75%) were captured using light traps and 368 (2.25%) by ad hoc hand catches (Table 3). Eighteen species repre- senting seven genera were identified morpho- logically: Anopheles (Anopheles) claviger (Meigen), An. (Ano.) hyrcanus (Pallas), An. (Ano.) maculipennis Meigen s.l., An. (Ano.) pseudopictus Grassi, An. (Cellia) superpictus, Aedes (Aedimorphus) vexans (Meigen) [Ae- dimorphus vexans], Ae. (Dahliana) genicula- tus (Olivier) [Dahliana geniculata], Ae. (Och- lerotatus) caspius s.l. [Ochlerotatus caspius s.l.], Ae. (Och.) pulcritarsis (Rondani) [Oc. pul- critarsis], Coquillettidia (Coquillettidia) richi- ardii (Ficalbi), Cx. (Culex) mimeticus Noè, Cx. (Cux.) perexiguus Theobald, Cx. (Cux.) pipiens, Cx. (Cux.) theileri, Cx. (Cux.) tritaeniorhynchus Giles, Culiseta (Culiseta) annulata (Schrank), Orthopodomyia pulcripalpis and Uranotaenia (Pseudoficalbia) unguiculata Edwards (Table 3). Aedes pulcritarsis and Cx. perexiguus were found for the first time in Guilan Province. Species dominance structure Overall, 2734 anopheline adults (16.71%) and 13,593 culicine adults (83.29%) were col- lected. The most prevalent species were Cx. theileri (23.59%, dominant), Cx. tritaeniorhyn- chus (20.75%, dominant), Cx. pipiens (19.37%, dominant), Ae. vexans (18.18%, dominant), An. J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 401 http://jad.tums.ac.ir Published Online: December 25, 2018 pseudopictus (10.92%, dominant) and An. mac- ulipennis s.l. (5.48%, subdominant). These six species included 16,054 specimens (98.1%) of the whole sample (Table 3). Moreover, they showed the widest distributions in the province (Table 4, Fig. 2). Regarding the dominance structure of subfamily Anophelinae, An. pseu- dopictus and An. maculipennis s.l. with the abundance percentages of 65.2% and 32.8% respectively, were both eudominant. In the case of percentage representation of subfamily Cu- licinae, Cx. theileri (28.34%), Cx. tritaeniorhyn- chus (24.93%), Cx. pipiens (23.27%) and Ae. vexans (21.84%) were dominant, as in the case of all mosquitoes, i.e. the total for both sub- families. Seasonal activity and the fluctuations of rainfall and temperature In general, the active season of adult mos- quitoes extended from early May to early Oct in the fixed site (Pareh Village of Rudbar Coun- ty). The peak of activity was late June for Cx. theileri, mid-July for An. maculipennis s.l., An. pseudopictus and Cx. pipiens, and late July for Cx. tritaeniorhynchus. While the peak of activity of most adult mosquitoes was late June to mid-July, and the abundance dramat- ically decreased after that, the monthly mean temperature increased by Aug. Also after Apr rainfall decreased in the fixed site during Jun and Jul and the rainy season started in Sep (Figs. 3–6). There is no significant regression between the abundance of adult mosquitoes and the meteorological data (Table 2) during active season in the fixed site (P> 0.05, R2= 0.31). Table 1. Collection data for adult mosquitoes captures at variable sites in Guilan Province, Iran, August–December 2015 and April–October 2016 Locality (City/ Village) Topography County Coordinates Altitude (m) Rostamabad Plain Rudbar 36o 52.999' N, 49o 29.385' E 215 Joben Foothill Rudbar 36o 53.072' N, 49o 27.658' E 399 Khaskool Foothill Rudbar 36o 50.789' N, 49o 32.669' E 470 Lafandsara Foothill Rudbar 36o 50.522' N, 49o 32.271' E 620 Rudbar Foothill Rudbar 36o 49.314' N, 49o 25.322' E 270 Klayeh Foothill Rudbar 36o 50.992' N, 49o 32.132' E 438 Rudabad Plain Rudbar 36o 52.397' N, 49o 30.871' E 192 Harkian Foothill Rudbar 36o 59.592' N, 49o 33.491' E 149 Siahroodposhteh Foothill Rudbar 36o 59.862' N, 49o 33.432' E 269 Upper Harzavil (Manjil) Foothill Rudbar 36o 44.495' N, 49o 26.072' E 506 Lower Harzavil (Manjil) Foothill Rudbar 36o 44.837' N, 49o 25.735' E 453 Halaj (Loshan) Foothill Rudbar 36o 40.306' N, 49o 26.792' E 307 Kacha Foothill Rasht 37o 05.173' N, 49o 36.973' E 124 Saghalaksar Plain Rasht 37o 09.596' N, 49o 31.334' E 53 Ghazian Coastal Anzali 37o 27.347' N, 49o 28.663' E -21 Kandbon Plain Rudsar 37o 03.415' N, 50o 20.987' E 20 Kalesara Foothill Talish 37o 42.251' N, 48o 55.577' E 93 Eivazmahaleh Foothill Astara 38o 23.964' N, 48o 46.715' E 77 Sechekeh Foothill Siahkal 37o 06.755' N, 49o 50.985' E 205 Asooyebala (Tootaki) Mountainous Siahkal 37o 03.556' N, 49o 52.542' E 355 J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 402 http://jad.tums.ac.ir Published Online: December 25, 2018 Table 2. The meteorological data of the fixed site (Pareh Village of Rudbar County), Guilan Province, Iran, 2016 Meteorological data April May June July Aug September October November Maximum Temperature 20.88 26.71 30.00 29.68 33.53 27.94 20.64 14.91 Minimum Temperature 10.79 16.56 20.17 21.52 22.60 18.77 12.92 6.25 Average Temperature 15.84 21.64 25.08 25.60 28.06 23.36 16.78 10.58 Relative Humidity 69.06 67.04 59.58 65.86 56.53 67.63 72.65 67.39 Rainfall 40.34 17.89 10.73 36.95 2.22 56.71 58.87 73.30 Fig. 1. Map of Iran highlighting the location of Guilan Province including mosquito collection sites surveyed in 2015–2016 J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 403 http://jad.tums.ac.ir Published Online: December 25, 2018 Table 3. The collection method and composition of adult mosquitoes collected in Guilan Province, Iran, August– December 2015 and April–October 2016 Species Light trap Hand catch Total Dominance structure n % n % n % An. claviger 18 0.11 2 0.54 20 0.12 Subrecedent An. hyrcanus 29 0.18 - - 29 0.17 Subrecedent An. maculipennis s.l. 752 4.71 144 39.13 896 5.48 Subdominant An. pseudopictus 1760 11.03 23 6.25 1783 10.92 Dominant An. superpictus 6 0.04 - - 6 0.04 Subrecedent Ae. caspius s.l. 96 0.60 - - 96 0.60 Subrecedent Ae. geniculatus 1 0.01 - - 1 0.01 Subrecedent Ae. pulcritarsis 7 0.04 - - 7 0.04 Subrecedent Ae. vexans 2930 18.36 39 10.59 2969 18.18 Dominant Cq. richiardii 94 0.59 1 0.28 95 0.60 Subrecedent Cx. mimeticus 8 0.05 1 0.28 9 0.06 Subrecedent Cx. perexiguus 6 0.04 - - 6 0.04 Subrecedent Cx. pipiens 3030 18.98 134 36.41 3164 19.37 Dominant Cx. theileri 3844 24.09 9 2.44 3853 23.59 Dominant Cx. tritaeniorhynchus 3375 21.15 14 3.80 3389 20.75 Dominant Cs. annulata 1 0.01 - - 1 0.01 Subrecedent Or. pulcripalpis - - 1 0.28 1 0.01 Subrecedent Ur. unguiculata 2 0.01 - - 2 0.01 Subrecedent Total 15959 100 368 100 16327 100 Table 4. The distribution of adult mosquitoes collected in different counties in Guilan Province, Iran, August– December 2015 and April–October 2016 Locality Species County Rudbar Rasht Anzali Rudsar Talish Astara Siahkal An. claviger * - - - - - - An. hyrcanus - * - * - - - An. maculipennis s.l. * * * * * - * An. pseudopictus * * * * * - * An. superpictus * - - - - - - Ae. caspius s.l. * - * - - - - Ae. geniculatus * - - - - - - Ae. pulcritarsis * - - - - - * Ae. vexans * * * * - - * Cq. richiardii - - * - - - - Cx. mimeticus * - - * - - - Cx. perexiguus * - - - - - - Cx. pipiens * * * * * * * Cx. theileri * * * - - - * Cx. tritaeniorhynchus * * * * * * * Cs. annulata - * - - - - - Or. pulcripalpis * - - - - - - Ur. unguiculata * - - - - - - J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 404 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 2. Distribution map of the most prevalent and medically important mosquitoes in the study areas in Guilan Province, Iran, 2015–2016 Fig. 3. Biweekly abundance of the most prevalent anopheline mosquitoes and monthly mean temperature in the fixed site, Pareh Village of Rudbar County, Guilan Province, Iran, April–October 2016 (Total includes Anopheles claviger, An. maculipennis s.l., An. pseudopictus, An. superpictus, Ae. pulcritarsis, Culex mimeticus, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus) J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 405 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 4. Biweekly abundance of the most prevalent anopheline mosquitoes and monthly rainfall in the fixed site, Pareh Village of Rudbar County, Guilan Province, Iran, April–October 2016 (Total includes Anopheles claviger, An. maculipennis s.l., An. pseudopictus, An. superpictus, Ae. pulcritarsis, Culex mimeticus, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus) Fig. 5. Biweekly abundance of the most prevalent culicine mosquitoes and monthly mean temperature in the fixed site, Pareh Village of Rudbar County, Guilan Province, Iran, April–October 2016 (Total includes Anopheles claviger, An. maculipennis s.l., An. pseudopictus, An. superpictus, Ae. pulcritarsis, Culex mimeticus, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus) J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 406 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 6. Biweekly abundance of the most prevalent culicine mosquitoes and monthly rainfall in the fixed site, Pareh Village of Rudbar County, Guilan Province, Iran, April–October 2016 (Total includes Anopheles claviger, An. maculipennis s.l., An. pseudopictus, An. superpictus, Ae. pulcritarsis, Culex mimeticus, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus) Discussion Mosquito fauna During the present investigation, 18 species representing seven genera of mosquitoes were collected in Guilan Province in which Ae. pul- critarsis and Cx. perexiguus were new records for the province. Thus, the number of species recorded in the province increased from 30 (36) to 33 (35 and the present investigation). Seven species, An. (Ano.) plumbeus Ste- phens, Ae. (Dah.) echinus (Edwards) [Dahliana echinus], Cx. (Maillotia) hortensis Ficalbi, Cx. (Cux.) torrentium Martini, Cx. (Neoculex) ter- ritans Walker, Cs. (Allotheobaldia) longiare- olata (Macquart) and Cs. (Culicella) morsitans (Theobald) whose larvae were collected during recent years in Guilan Province (36), were not captured in the present study. The reason is the rarity of some aforementioned species and the tendency of some, such as Cx. hortensis, Cx. territans and Cs. morsitans, to feed on birds, amphibians or reptiles (54), thus they were not attracted to light traps used in animal (cattle and sheep) shelters during the study. Also, there are seven species of the Maculi- pennis Group in Guilan Province differenti- ated by egg patterns or the polymerase chain reaction (PCR) technique (36), which could not be differentiated with the morphology- based keys of females and larvae (29) used in the present study. Among the species collected, Ae. caspius s.l. is known to vector WNV in West Azer- baijan Province in northwestern Iran (14). Shahhosseini et al. (55) referred to the virus lat- er as Kunjin-related West Nile Virus. More- over, WNV was found in Cx. pipiens in the Sepid-Rud valley of Guilan Province (15). In addition, An. maculipennis s.l., Ae. vexans, Cq. richiardii, Cx. perexiguus, Cx. theileri, Cx. tritaeniorhynchus and Ur. unguiculata is be- lieved to play role as vectors of WNV in dif- ferent countries of the Old World (1, 56). Among the aforementioned species, Cq. richi- ardii and Cx. pipiens in Europe and Cx. tri- taeniorhynchus in Asia are the main vectors of J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 407 http://jad.tums.ac.ir Published Online: December 25, 2018 the virus (1). Culex theileri has been found to be the vec- tor of Dirofilaria, the causal agent of diro- filariasis, in Ardebil Province in northwestern Iran (27), and An. claviger, An. hyrcanus, An. maculipennis, An. pseudopictus, An. superpic- tus, Ae. vexans, Ae. caspius, Ae. geniculatus, Cq. richiardii, Cx. pipiens, Cx. tritaeniorhyn- chus, Cs. annulata and Ur. unguiculata are known the vectors of Dirofilaria in different countries of the western Palaearctic Region (54, 57–59). Species dominance structure In the present study, the most abundant species were Cx. theileri, Cx. tritaeniorhynchus, Cx. pipiens, Ae. vexans, An. pseudopictus and An. maculipennis s.l., respectively. With the exception of An. maculipennis s.l., which is subdominant, they are all dominant according to the classification (52, 53) (Table 3). This is concordant with the previous findings in the province based on collections of larvae (36– 38), as well as adults (37, 60). The exception is Cx. theileri. The species had been found less often in the larval stage than any other Culex in the province (38). One reason is probably due to sampling, the heavy rainfall in the prov- ince results in a great number of different nat- ural larval habitats that are favorable for Cx. theileri (38), but those habitats are not easily located and sampled. On the other hand, the favorable larval habitats of some species, such as Cx. pipiens and Cx. tritaeniorhynchus, i.e. artificial containers and rice fields, respective- ly, are easier to find and sample. Another rea- son is probably the biology of the species, some species such as Cx. hortensis and Cx. territans, which do not bite humans and mammals but mostly feed on amphibians, reptiles or birds (54), have been collected very often as larvae (38). They were not collected during the pre- sent investigation by means of aspirators and light traps from animal shelters which attract Cx. theileri (Table 3). The most prevalent spe- cies of the province, An. maculipennis s.l., An. pseudopictus, Ae. vexans, Cx. pipiens, Cx. theil- eri and Cx. tritaeniorhynchus are known vec- tors of both WNV and Dirofilaria (1, 57, 59). Seasonal activity and the fluctuations of rainfall and temperature During the present investigation, Ae. vexans was one of most abundant species and most prevalent aedine species (Table 3), as noted previously (37, 60). However, most specimens were collected from Anzali (Table 4) and the species was not collected from the fixed site, so its seasonal activity is not discussed here. Anopheles maculipennis s.l. showed the peak of activity in the mid-Jul (Figs. 3, 4). The peak of monthly activity of anophelines (including An. maculipennis s.l. and An. superpictus) was reported during Jul–Aug in Kalaleh County of Golestan Province, northern Iran (47). The most An. maculipennis s.l. was captured in Aug in Aras Valley, Turkey, adjoining Iran (61). There are no records for the seasonal activity of cu- licine adults in Iran. The peak of activity of Cx. pipiens was found in Jul in northern Italy (62). That is in concordance with the result of the present study (Figs. 5, 6). However, the peak of activity of Cx. pipiens was recorded in Aug in the Belek Region and Aras Valley of Tur- key (61, 63). Most Cx. theileri was found in Jun in Aras Valley of Turkey (61), which is similar to the present study (Figs. 5, 6), how- ever, the peak activity of this species was re- ported in Aug in Ankara, Turkey (64). More- over, most Cx. tritaeniorhynchus was captured in Aug in Belek Region of Turkey (63), while the peak of activity was observed in Jul in the present study (Figs. 5, 6). Differences between the results of the present investigation and the findings in other regions may be explained by differences in the topography and climates (es- pecially temperature) which influence the bio- nomics of mosquitoes. On the other hand, some differences are due to sampling regimes. For example, mosquito abundance was reported based on weekly catches (62), and on month- ly catches (61, 63, 64), whereas the seasonal J Arthropod-Borne Dis, December 2018, 12(4): 398–413 Sh Azari-Hamidian et al.: Seasonal Activity of … 408 http://jad.tums.ac.ir Published Online: December 25, 2018 activity is based on biweekly captures in the present study and another study (47). Besides, dry-ice baited traps was used for sampling by Alten et al. (63), BG-traps by Roiz et al. (62) and spray sheet collections by Sofizadeh et al. (47), whereas light traps were used by Simsek (64), Alkan and Aldemir (61) and in this study. Though the mean monthly temperature of about 16 °C is a limiting factor in the activity of adult mosquitoes in the study area (Figs. 3, 5), no significant regression was observed between different meteorological data (Table 2) and the abundance of adult mosquitoes dur- ing active season (P> 0.05, R2= 0.31). During the present study, the peak of activity of most adult mosquitoes was late Jun to mid-Jul, only the peak of activity of Cx. tritaeniorhynchus was in late Jul (Figs. 3–6), after which the abun- dance dramatically decreased as temperature increased by Aug (Figs. 3, 5). High tempera- ture (> 35 °C) is generally a limiting factor for the abundance of adult mosquitoes, especially in localities with warm climate such as south- ern Iran (41) and Saudi Arabia (65). However, it does not seem that temperature was a key factor in decreasing the abundance of adult mosquitoes in the area of the present study, because the temperature does not exceed 34 °C and the monthly mean temperature is lower than 30 °C in the fixed site in Aug (Figs. 3, 5). In addition, rainfall decreased in the fixed site during Jun and Jul while the abundance in- creased. The rainy season started in Sep while the abundance of mosquitoes dramatically de- creased (Figs. 4, 6). A key factor mentioned here is rice fields, the main larval habitats of the most prevalent species, are dry during Aug. Temperature decreases significantly during Sep and Oct, consequently, the prevalence of mos- quitoes decreases. In view of integrated vector management, ecological data, especially seasonal activity, is very important for intervention measurements. On the other hand, one of main intervention measurements is using pesticides yet. There is little-published data about the susceptibility sta- tus of mosquitoes, especially culicines (66, 67), in northern Iran. This subject can be a goal for forthcoming studies in Guilan Province. Conclusion Though there was no significant regression between the abundance of adult mosquitoes and the meteorological data in the fixed site during active season, temperature and rice fields had a great influence in starting and ending active season in the region. The seasonal activity of the important species Ae. vexans, other species found less abundant in this study, host pref- erence analysis and filarial and arbovirus screen- ing should be the subjects of future investi- gations in the region. Acknowledgements The authors appreciate the administrative support of Dr SM Rezvani, Mr M Mirzanejad, Mr M Moslem and Mr A Rasaei, Department of Disease Control and Prevention, Health Vice-Chancellorship of Guilan University of Medical Sciences, Rasht, Iran, and the per- sonnel of aforementioned vice-chancellorship for their cooperation in the field. Ms R Far- hadi, and Mr Omidi, School of Health, Gui- lan University of Medical Sciences, are ap- preciated for helping in providing the graphs of seasonal activities and data analysis, re- spectively. The authors are grateful to Dr RE Harbach, Department of Life Sciences, Natu- ral History Museum, London, UK, for review- ing the manuscript. The research was supported financially by the Research Vice-Chancellorship of Guilan University of Medical Sciences (Project Num- ber: 93062502). 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