J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 240 http://jad.tums.ac.ir Published Online: September 30, 2018 Original Article Fauna and Larval Habitats Characteristics of Mosquitoes (Diptera: Culicidae) in Golestan Province, Northeast of Iran, 2014–2015 *Aioub Sofizadeh 1, Hamid Reza Shoraka 2, Fatemeh Mesgarian 3, Ghorban Mohammad Ozbaki 3, Abdolsamad Gharaninia 3, Ebrahim Sahneh 3, Rohollah Dankoob 3, Ali Malaka 3, Saeid Fallah 3, Shamsaddin Nemani 3 1Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran 2North Khorasan University of Medical Scinces, Bojnurd, Iran 3Health Centers of Health Deputy, Golestan University of Medical Sciences, Gorgan, Iran (Received 30 July 2017; accepted 11 June 2018) Abstract Background: Mosquitoes (Diptera: Culicidae) is one of the most medically important families of Diptera. The aims of this study were to determine fauna and larval habitat characteristics of mosquitoes in Golestan Province, during 2014–15. Methods: This study was conducted in larval habitats of mosquitoes and installed ovitraps in 14 districts of Golestan Province, Northern Iran in 2015. Samples were collected with a scoop by ladle handling for entomology. The col- lected larvae were transferred to Laboratory of Medical Entomology in lactophenol solution. Then microscopic slides were prepared using de Faure's formula. Species of each sample was recognized using diagnostic criteria to identify the Culicidae species. Characteristics of larval breeding places were studied based on the habitat type (Permanent or temporary), water conditions (Clear or turbid, stagnant or running), vegetation (out, in, underwater vegetation or without vegetation), sunlight exposure (Full or partial sunlight) and so on. Data were analyzed using SPSS. Results: Overall, 5661 third- and fourth- instars larvae of mosquitoes were collected and 5 genera and 14 species were identified: Anopheles hyrcanus, An. maculipennis, An. pseudopictus, An. superpictus, Culex hortensis, Cx. mimiticus, Cx. perexiguus, Cx. pipiens, Cx. pusillus, Cx. theileri, Cx. tritaeniohynchus, Culiseta longiareolata, Och- lerotatus caspius, Uranotaenia unguiculata. Culex pipiens was recognized as predominant species of the family. Among the detected species, Cx. pusillus reported for the first time from Golestan Province. Conclusion: Due to the high species diversity of Culicidae, ecology of medical important species such as Cx. pipiens and Cx. tritaeniorhynchus needs more investigations. Keywords: Culicidae, Larval habitat, Ecology, Iran Introduction Mosquitoes (Diptera: Culicidae) as one of the most important families of insects. Ac- cording to the latest taxonomy, Culicidae com- prises two subfamilies, 11 tribes, 112 genera and 3539 species (1). The checklist of the mosquitoes in Iran includes 7 genera, 16 sub- genera, 64 species and three subspecies (2). Subsequently, Anopheles superpictus is two species in Iran based on the Internal Tran- scribed Spacer 2 (ITS2) sequences of rDNA (3), later listed as species A and B (4). A new species of the Anopheles hyrcanus group (An. hyrcanus spIR) was recognized from south- western Iran also based on ITS2 sequence data (5). More recently, the occurrence of Ae- des albopictus was reported in southeastern Iran and Orthopodomyia pulcripalpis in north- ern Iran, respectively (6, 7). The fauna of mosquitoes of Golestan Prov- ince, Northern Iran includes Aedes (Aedi- morphus) vexans, Ae. (Dahliana) echinus, Ae. (Dah.) geniculatus, Ae. (Ochlerotatus) caspius, *Corresponding author: Dr Aioub Sofizadeh, E- mail: a_sofizadeh@yahoo.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 241 http://jad.tums.ac.ir Published Online: September 30, 2018 Ae. (Och.) pulcritarsis, Anopheles alrgeriensis, An. claviger, An. hyrcanus, Anopheles. mac- ulipennis s.l., An. melanoon, An. multicolor, An. plumbeus, An. pseudopictus, An. pulcher- rimus, An. superpictus, Coquillettidia richi- ardii, Culex hortensis, Cx. mimeticus, Cx. perexiguus, Culex. pipiens, Cx. territans, Cx. theileri, Culiseta longiareolata, Cs. subochrea, Uranotaenia unguiculata (8–13). Mosquitoes are important vectors of many diseases in- cluding: malaria, West Nile virus, dengue fe- ver, yellow fever, filariasis and other diseas- es (14). Some of these diseases and their agents have previously been reported from Golestan Province, for example: malaria, West Nile Virus, Dirofilaria immitis (15-17). Malaria as one of the most important vector-borne diseases in Iran is transmitted by Anopheles mosquitoes. Over the past dec- ades, north of the country including Golestan Province has been identified as one of the most important endemic foci of malaria (15), eight species of the genera Anopheles are known as vectors of malaria in Iran. An. culicifacies s.l., An. dthali, An. fluviatilis s.l., An. maculipennis s.l., An. sacharovi, An. ste- phensi, and An. superpictus have been intro- duced as primary and secondary malaria vec- tors and An. pulcherrimus as a suspected vec- tor (11). West Nile virus is a mosquito-borne virus that transmitted by Culex species to birds, equines, and humans (18) seropositive cases of humans and equines for these virus- es were reported in Golestan Province (16). Dirofilaria immitis also is one of the most important mosquito-borne pathogens that re- ported in Golestan Province (17), Culex theil- eri is known vector of this pathogen in the north-west of Iran (19). Mosquitoes play a key role in transmit- ting diseases, many have studies carried out in north of Iran and Golestan Province on the biology and ecology of mosquitoes: larval habitats characteristics of mosquitoes and bi- ological characteristics of Anopheles was re- ported in Kalaleh County, Golestan Province (12, 13). Fauna and larval habitats of mosqui- toes were reported in Guilan and North Khora- san Provinces, north of Iran (20-23). Fauna, larval habitat and other biological character- istics of mosquitoes were reported in Ma- zandaran Province and Neka County (24-26). Physicochemical characteristics of mosqui- toes were studied in Qom Province (27) and larval habitats, affinity and diversity indices of Culicinae in Southern Iran (28). Different species of mosquitoes require different bio- logical and ecological conditions to growth and development. While some lay eggs and breed in permanent water habitats, others pre- fer temporary water bodies for breeding, some favour feeding on sweet water, others need salty water, and some prefer high tempera- ture and humidity while others are in favour of low temperature and humidity. Regardless of these differences, is clear and evident that all need water for breeding and without wa- ter, their chances for growth and develop- ment would be slight. Though the climate in Golestan Province is suitable for the growth and development of mosquitoes such as Anopheles, Aedes and Culex mosquitoes, so far, no comprehensive studies have been carried out on the fauna and ecological properties of mosquitoes in Golestan Province. To address this gap, this research was an attempt to analyze the char- acteristics of mosquito larval habitats. Materials and Methods Study area Golestan Province (53°57′–56°23′ E, 36° 30′–38°08′ N) covering a landmass of 20437.74 square km consists of approximately 1.3% of the total area of Iran. It is located in the north- eastern region of the country and bordered by the Republic of Turkmenistan to the north, Alborz Mountain range and Semnan Prov- ince to the south, North Khorasan Province to the east, and Caspian Sea and Mazandaran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 242 http://jad.tums.ac.ir Published Online: September 30, 2018 Province to the west. This province consists of 14 counties, 25 cities, 60 districts and 1764 villages (Fig. 1). The province is enriched with diverse ecology and climatic conditions. Con- sidering the sea, forest and mountainous areas, the climatic condition of the province is clas- sified into temperate mountainous, cold moun- tainous (3000m high), a mild Mediterranean and arid and semi-arid regions, such that as we move from southern to northern parts, the amount of rainfall and relative humidity de- creases and the degree of temperature increas- es. With regards to the topography of the prov- ince, this region is subdivided into three dis- tinct areas; mountainous, plain and even posts. The mountainous areas are located in the southern parts containing the highest peaks of the province. Mountainous areas are located in the foothills of the southern and eastern borders of the province with coarse sediments as alluvial fans make use of this land. As a result of the high permeability of the soil in the mountainous areas, groundwater aquifers with water in wells and canals are exploited. The retreat of the Caspian Sea post and plain regions has created severe water erosion and compaction of alluvial rivers. The lowest parts of the province (around the Caspian Sea with an altitude of 32m above sea level) are located in an area inhabited by a majority of the province's population (29). Specimen and data collection This study was performed in all counties of Golestan Province, northern Iran in 2015. In each county, one city and two villages were selected based on the topographic conditions. In order to sampling of larves, in each select- ed city and villages, at least two larval habi- tats were searched. First, features such as lar- val habitat status (permanent or temporary, stagnant or slow-running water), vegetation type, substrate type, habitat types and posi- tion of the sunlight were recorded on special forms. Then, larval sampling method was car- ried out using standard dipper of 350ml. Each habitat was sampled in different parts of the larval habitats for 10 times. All captured lar- vae in each dipper were counted and collect- ed in special containers. The information of habitats was recorded on the larva containers and transferred to the medical entomology la- boratory in Health Center of Kalaleh County. In the laboratory, after drying, larvae were kept in lactophenol medium and were mounted on microscope slides in de Faure's formula and using standard taxonomic keys (30), larvae species were determined. Sampling was done once in each of the seasons of spring, sum- mer, and autumn. Preparation of Ovitrap and larva sampling of this trap for searching Aedes eggs and larva We used CDC Ovitrap. This consists of a three-liter, black, water-filled, plastic container and thin paddle of wood (2×12.5cm) placed in the container (31, 32). In each selected city and village, 10 ovitraps were implemented and investigated once a week and collected the present larvae and as it was explained previ- ously, they were mounted and Species of each sample was recognized. Results Overall, 5661 larvae belonged to Culicidae were collected and their species were identi- fied. Including An. hyrcanus, An. maculipennis s.l., An. pseudopictus, An. superpictus s.l., Cx. hortensis, Cx. mimeticus, Cx. perexiguus, Cx. pipiens, Cx. pusillus, Cx. theileri, Cx. tri- taeniohynchus, Cs. longiareolata, Oc. caspius, and Uranotaenia unguiculata. Of 2821 ones had been collected from natural larval habitats and 2840 ones from the prepared ovitraps (Table 1). Culex pipiens was identified as the dominant species of Golestan Province and it was collected from all counties of this province. In terms of fre- quency, Cx. tritaeniohynchus was in the sec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 243 http://jad.tums.ac.ir Published Online: September 30, 2018 ond rank and was collected from a majority of counties (Table 2). In the present study, 77.3% of larvae were collected from tempo- rary larval habitats compared to permanent ones and 73.6% of larvae from larval habi- tats with stagnant water compared to those with running water (Table 3). More larvae were collected from natural larval habitats of wetlands (31.3%) and artificial larval habi- tats of bogs (26.4%) (Table 4, 5). Table 1. Abundance of mosquito (Culicidae) larvae in Golestan Province, Nortehrn Iran 2015 species Natural larval habitats Ovitrap Total Num. % Num. % Num. % An. hyrcanus 2 0.1 0 0 2 0.001 An. maculipennis 20 0.7 0 0 20 0.4 An. pseudopictus 14 0.5 0 0 14 0.2 An. superpictus 178 6.3 0 0 178 3.1 Cx. hortensis 6 0.2 0 0 6 0.1 Cx. mimeticus 46 1.6 2 0.1 48 0.8 Cx. perexiguus 72 2.5 7 0.2 79 1.4 Cx. pipiens 1657 58.2 2736 96.3 4393 77.3 Cx. pusillus 1 0.001 3 0.1 4 0.1 Cx. theileri 5 0.2 4 0.1 9 0.2 Cx. tritaeniohynchus 617 21.7 88 3.1 705 12.4 Cs. longiareolata 18 0.6 0 0 18 0.3 Oc. caspius 183 6.4 0 0 183 3.2 Uranotaenia unguiculata 2 0.1 0 0 2 0.001 Total 2821 100 2840 100 5661 100 %,%, %, %, %, %, %, %, %, %, %, %, %, %, %, %, %, %,%, %, %, %, %, %, %, %, %, %,%, %, %, %, %, %, %,%, %,%, %,%, %, %,%, Gonbad-e Kavoos Kalaleh Gorgan Aghghala Maraveh Tappeh Gamishan Galikesh Ramiyan Kordkooy Azadshahr Aliabad-e Katool Minoodasht Bandar-e Gaz Bandar-e Torkaman ® Legend %, selected villages for sampling golestan counties 0 25 50 75 10012.5 Kilometers Fig. 1. Location of Golestan Province and selected villages for sampling of Culicidae http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 244 Table 2. Distribution of mosquitoes in different counties of Golestan Province, northern Iran Counties Species M a r a v e h T a p e h K a la le h G o n b a d -e K a v u s G a lik e sh M in o o d a sh t A z a d sh a h r R a m ia n A lia b a d -e K a tu l A q Q a la G o r g a n K o r d k o u y B a n d a r -e G a z B a n d a r -e T u r k m a n G o m ish a n T o ta l An. hyrcanus 0 0 0 0 2 0 0 0 0 0 0 0 0 0 2 An. maculipennis 0 15 0 5 0 0 0 0 0 0 0 0 0 0 20 An. pseudopictus 0 0 0 0 14 0 0 0 0 0 0 0 0 0 14 An. superpictus 0 178 0 0 0 0 0 0 0 0 0 0 0 0 178 Cx. hortensis 0 0 0 6 0 0 0 0 0 0 0 0 0 0 6 Cx. mimeticus 0 5 0 18 24 0 0 1 0 0 0 0 0 0 48 Cx. perexiguus 63 1 0 8 3 0 0 0 0 0 0 3 1 0 79 Cx. pipiens 164 152 1658 60 156 223 161 564 40 80 742 265 74 54 4393 Cx. pusillus 3 1 0 0 0 0 0 0 0 0 0 0 0 0 4 Cx. theileri 4 0 3 0 0 0 0 0 0 0 0 2 0 0 9 Cx. tritaeniohynchus 53 62 221 103 59 0 5 49 4 0 5 59 85 0 705 Cs. longiareolata 0 18 0 0 0 0 0 0 0 0 0 0 0 0 18 Oc. caspius 0 0 104 0 0 0 0 28 0 0 0 0 51 0 183 Uranotaenia unguiculata 0 1 0 0 0 0 0 0 0 0 0 0 1 0 2 Total 287 433 1986 200 258 223 166 642 44 104 747 329 212 54 5661 Table 3. Larval habitat characteristics of mosquitoes collected in Golestan Province, northern Iran 2015 Species Larval habitats characteristics A n . h y rc a n u s A n . m a c u lip e n n is A n . p se u d o p ic tu s A n . su p e rp ic tu s C x . h o rte n sis C x . m im e tic u s C x . p e re x ig u u s C x . p ip ie n s C x . p u sillu s C x . th e ile ri C x . trita e n io h y n c h u s C s. lo n g ia re o la ta O c . c a sp iu s U ra n o ta e n ia u n g u ic u la ta T o ta l Habitat situation Permanent 2 0 7 29 4 2 33 488 1 0 56 0 25 0 645 Temporary 0 20 7 148 4 44 39 1169 0 5 561 18 158 20 2193 running water 2 0 7 83 4 2 8 523 1 2 118 0 0 0 750 Stagnant water 0 20 7 95 2 44 64 1134 0 3 499 18 183 20 2089 Vegetation situation Out of water 0 0 0 121 0 34 56 527 0 5 282 0 104 0 1165 In water level 0 0 0 0 4 12 6 370 0 0 136 0 28 0 571 underwater 0 0 0 0 0 0 8 49 0 0 2 0 26 0 85 without 0 0 0 57 2 0 2 711 1 0 197 18 25 0 1013 Sunlight situation Full sunlight 0 0 0 28 0 5 30 537 0 5 389 0 127 0 1124 Shaded 0 0 0 0 0 0 0 64 0 0 20 0 30 0 118 Partial sunlight 2 5 7 26 0 19 38 399 0 0 101 0 10 0 607 Sunlight shaded 0 15 7 124 0 22 4 657 1 0 110 18 16 1 975 Substrate mud 2 19 14 149 6 46 72 1004 0 5 481 0 158 2 1958 sand 0 1 0 29 0 0 0 279 1 0 136 0 25 0 471 rock 0 0 0 0 0 0 0 70 0 0 0 0 0 0 70 others 0 0 0 0 0 0 0 295 0 0 0 18 0 0 313 Water Situation J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 245 muddy 0 0 0 28 0 0 5 372 0 0 48 0 0 0 453 clear 2 20 14 150 6 46 66 1114 1 4 497 18 155 1 2094 Turbid 0 0 0 0 0 0 1 171 0 1 72 0 28 1 274 fresh 2 20 14 95 6 46 29 1571 0 5 494 18 158 1 2459 salty 0 0 0 0 0 0 26 34 0 0 18 0 0 0 78 brackish 0 0 0 83 0 0 17 52 1 0 105 0 25 1 284 Habitat Kind Natural 2 5 14 83 6 41 28 446 1 4 310 2 183 1 1126 Artificial 0 15 0 95 0 5 44 1211 0 1 307 16 0 1 1695 Table 4. Abundance of species of mosquitoes in natural habitats in Golestan Province, northern Iran, 2015 Natural habitats Type Species R iv e r E d g e R iv e r b e d M a r sh C r e e k F o u n ta in P it W e tla n d s T r e e h o le s W a te r le a k a g e T o ta l An. hyrcanus 2 0 0 0 0 0 0 0 0 2 An. maculipennis 0 0 0 0 0 0 5 0 0 5 An. pseudopictus 7 0 7 0 0 0 0 0 0 14 An. superpictus 29 0 0 26 0 0 28 0 0 83 Cx. hortensis 0 2 0 4 0 0 0 0 0 6 Cx. mimeticus 2 0 10 0 0 7 5 0 17 41 Cx. perexiguus 1 0 0 8 6 2 8 0 3 28 Cx. pipiens 9 9 13 52 0 109 144 0 37 446 Cx. pusillus 1 0 0 0 0 0 0 0 0 1 Cx. theileri 0 0 0 2 0 2 0 0 0 4 Cx. tritaeniohynchus 73 2 16 9 2 10 124 0 74 310 Cs. longiareolata 0 0 0 0 0 0 0 2 0 2 Oc. caspius 0 0 25 104 0 16 38 0 0 183 Uranotaenia unguiculata 0 0 0 0 0 0 1 0 0 1 Total 197 13 71 205 8 146 353 2 131 1126 Table 5. Abundance of species of Culicidae in artificial habitats in Golestan Province, northern Iran, 2015 Artificial habitats type species C r e a te sid e s o f r ic e C r e a te m id d le o f r ic e S tr e a m o f c r e a tin g r ic e B o g O th e r a g r ic u ltu r a l str e a m s C iste r n L a k e W e ll P o o l O th e r s T o ta l An. hyrcanus 0 0 0 0 0 0 0 0 0 0 0 An. maculipennis 15 0 0 0 0 0 0 0 0 0 15 An. Pseudopictus 0 0 0 0 0 0 0 0 0 0 0 An. superpictus 95 0 0 0 0 0 0 0 0 0 95 Cx. hortensis 0 0 0 0 0 0 0 0 0 0 0 Cx. mimeticus 0 0 0 0 0 0 0 5 0 0 5 Cx. perexiguus 44 0 0 0 0 0 0 0 0 0 44 Cx. pipiens 114 25 57 446 153 0 48 0 94 274 1211 Cx. pusillus 0 0 0 0 0 0 0 0 0 0 0 Cx. theileri 1 0 0 0 0 0 0 0 0 0 1 Table 3. Continued … J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 246 Cx. tritaeniohynchus 33 12 48 1 199 0 0 9 0 5 307 Cs. longiareolata 0 0 0 0 0 16 0 0 0 0 16 Oc. caspius 0 0 0 0 0 0 0 0 0 0 0 Uranotaenia unguiculata 0 0 0 0 0 0 0 0 0 1 1 Total 302 37 105 447 352 16 48 14 94 280 1695 Discussion A total of 5661 larvae belonged to 5 gen- era and 14 species were identified: Anophe- les genus: 4 species, Culex genus: 7 species, Culiseta, Ochlerotatus, and Uranotaenia gen- era: one species. In previous studies of au- thors in Kalaleh County, An. hyrcanus, An. maculipennis s.l., An. pseudopictus, An. su- perpictus, Cx. hortensis, Cx. perexiguus, Cx. pipiens, Cx. theileri, Cs. Longiareolata and Oc. caspius had been reported (12, 13). Fur- thermore, species such as An. claviger and Cs. subochrea identified in Kalaleh study (13) were not collected in the present study. In previous studies of authors in Kalaleh County, Cx. pipiens, Cx. theileri, Cx. hortensis, Cx. perexiguus, An. maculipennis s.l., An. su- perpictus, An. hyrcanus, An. pseudopictus, Oc. caspius and Cs. longiareolata had been re- ported (12, 13). Furthermore, species such as An. claviger and Cs. subochrea identified in Kalaleh study (13) were not collected in the present study. In some further studies in north of Iran and Golestan Province related to the fauna and ecology of mosquitoes (8-13) were identified species such as: Aedes vexans, An. alrgeriensis, An. claviger, An. melanoon, An. multicolor, An. plumbeu, An. pulcherrimus, Coquillettidia richiardii, Cx. territans, Cs. sub- ochrea, Oc. echinus, Oc. geniculatus, Oc. pul- critarsis were not collected in our study, and Cx. pusillus were reported for the first time from Golestan Province. In the present study, the dominant species was Cx. pipiens and 77.3% of the whole col- lected larvae belonged to this species. In the studies of Mazandaran Province and Kalaleh County, (13, 25, 26), Moreover, Cx. pipiens had been reported as dominant species. The dominant species had been An. hyrchanus in Neka County, Mazandaran Province (24) and An. maculipennis and Cx. theileri in Ardebil Province in 2008 (19). Culex pipiens has been reported from almost all provinces of Iran (33). In the present study, too, this spe- cies was reported from all counties of Goles- tan Province. Larva habitats of this species varied, but mostly of samples were collected from Bogs, agricultural streams, wetlands and rice fields. In Mazandaran, reported wetlands and discarded tires the main larval habitats for Cx. pipiens (26). In Isfahan (34), this spe- cies was mostly found in rice fields and natu- ral habitats. Larval habitats of this species are mostly stagnant and artificial bodies of fresh water such as swamp of watering channels and holes and barrels filled with rain (35). Inves- tigation of Cx. pipiens larval habitats and their characteristics showed that this species was collected from different larval habitats with various ecological conditions; even, 96% of the whole collected species from ovitraps belonged to this species and this species has a very high adaptability with various larval habitats and this factor has caused its high frequency and distri- bution reported from Iran and the region (33). Culex tritaeniohynchus was one of the other species collected from a majority of counties and after Cx. pipiens, in the second rank, it in- cluded 12.4% of the whole collected larvae, 21.7% of larvae isolated from different lar- val habitats and also 3.1% of larvae isolated from ovitraps. Similar to our study, in Guilan, this species was in the second rank after Cx. pipiens (33), and in Mazandaran study (25), in the third rank after Cx. pipiens and Cx. torren- tium, while in Neka County of Mazandaran Table 5. Continued … J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 247 Province, Kurdistan, Esfahan and Qom Prov- inces studies, no larvae from this species has been collected (15, 36-38). In Golestan Prov- ince, this species has been collected from dif- ferent larval habitats, but its frequency in tem- porary and stagnant water habitats (95.7%) was more than that in permanent and running water habitats (4.3%). This observation is consistent with another study in Iran (21) but in Southern Iran (28), these species were collected in per- manent water higher than temporary water hab- itats. Furthermore, the frequency of this species in full sunlight situation and Mud substrate habitats was higher than in Shaded situation and sandy substrates habitats. In southern Iran (28), these species were collected in full sunlight and sandy substrate habitats higher than partial sunlight and mud substrate habitats. This spe- cies was mostly found in agricultural streams, wetlands, and water leakage and river edge. In this study, Cx. perexiguus was mostly found in Maraveh Tappeh County in the north- east of Golestan Province (80%), but it was found even in western counties of Golestan Province such as Bandar-e Gaz and Bandar-e Turkmen. These species prefers temporary lar- val habitats to permanent ones (13, 35, 39). In the present study, 55.5% of the collected lar- vae of this species were collected from tem- porary habitats. This species was only collect- ed from natural habitats and no larvae were collected from artificial habitats, In Kalaleh study (13) 89% larvae of this species were col- lected from natural habitats and in central Iran study (37) larvae of Cx. perexiguus were on- ly collected from rice fields as well in our study. The preferred habitats for this species were rice fields (61.1%). The notable point for habitats of this species is that it was collect- ed from habitats with sweet, salty and brack- ish water. This feature was also true for only Cx. pipiens and Cx. tritaeniohynchus species. Since this species was collected from ovitraps and habitats with different conditions, it has good adaptability with different conditions of larva habitats. From Culiseta genus, only Cs. logiareo- lata and from Kalaleh County was collected. The information about the ecology of Culiseta species in Iran is limited. In the present study, Cs. logiareolata was collected from only one larval habitat which was a destroyed cistern with cement substrate without vegetation. However, in Guilan Province, North Khora- san, Yazd and Esfahan provinces (20, 23, 34, 40) this species collected with high abun- dance and in other studies, this species had been collected from a variety of habitats (35, 36). In Yazd County, too, have reported Cs. logiareolata and Cx. pipiens in larval habi- tats infected with organic substances, indus- trial waste materials and cement pools for storing animals’ water (40). Anopheles maculipennis Group, An. clavi- ger, An. hyrcanus, An. plumbeus, An. alrger- iensis and An. multicolor and An. pulcher- rimus have been previously reported in Go- lestan Province (8-12), but in the present study, An. superpictus, An. maculipennis, An. psu- dopictus and An. hyrcanus were collected and identified. Anopheles superpictus and An. mac- ulipennis have been known as the vectors of malaria disease. In our study, An. superpictus was mostly found in the natural habitats of river beds and streams with permanent and stagnant water, also with or without vegeta- tion. These findings are completely similar to the results reported in previous studies (13, 36, 40). In Guilan Province, this species only reported of natural habitats with transi- ent, stagnant and clear water, and was mostly found in Rain pool habitats (23). The char- acteristics of larval habitats of An. superpic- tus are firstly shallow clear waters with stony bed and without vegetation and secondly riv- ers with sandy bed under sunlight and also shallow streams with muddy bed (34). More- over, in the present study, this species was collected from both types of the mentioned larval habitats. From genus Ochlerotatus, only Oc. caspius was collected, while in Kalaleh County study J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 248 (13), three species of these genera had been reported (Oc. caspius, Oc. echinus, and Oc. geniculatus). Ochlerotatus caspius has been collected in many studies in Iran (13, 27, 41– 43). In terms of frequency, in Kurdistan study (41), it was in the second rank after Cx. theileri. This high frequency of this species can be due to the Ochlerotatus mosquitos grow in humid forest regions and more selected vil- lages for sampling are in forest regions. In relation to the characteristics of larval habitats, Oc. caspius preferred temporary, running wa- ter, muddy Substrate and Full sunlight habi- tats. In Kurdistan Province (36) this species reported of river edge with stagnant and clear water and partial sunlight habitat. Although this species prefers larval habitats with clear and sweet water, it was also collected in lar- val habitats with turbid and brackish water. Culex mimeticus, Cx. theileri, Cx. hortensis, Cx. pusillus, An. maculipennis s.l., An. hyr- canus, An. pseudopictus and Uranotaenia un- guiculata were collected with low frequency in Golestan Province; the larval habitat char- acteristics of these species must be exten- sively studied. In addition, one of the aims of this study was to find Aedes genus larvae, but we did not collect any larvae of this genus in Goles- tan Province, although Ae. vexans was pre- viously reported from the province (8). Conclusion Due to the good climate conditions, dif- ferent species of mosquitoes grow in Goles- tan Province and since there are different vectors for various diseases among these species, conditions of disease transfer are present in this region. In the present study, we did not collect any species among the vectors of diseases such as dengue fever, Zika, and chikungunya. Future studies for finding these vectors in Golestan Province are recommended. Acknowledgements This research had been approved by The Infectious Diseases Research Center, Golestan University of Medical Sciences (35/72745). The authors would like to appreciate all staff of this center and Health centers of Golestan Province for their support and cooperation. Dr Azari-Hamidian, Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, is highly appreciated for his cooperation in the identification of species and reviewing the paper. References 1. Harbach RE (2007) Mosquito Taxonomic Inventory. The Natural History Muse- um, London, UK. Available at: http://mosquito-taxonomic- inventory. info/ 2. Azari-Hamidian S (2007) Checklist of Ira- nian mosquitoes (Diptera: Culicidae). J Vector Ecol. 32: 235–242. 3. Oshaghi MA, Yaghobi-Ershadi MR, Shemshad Kh, Pedram M, Amani H (2008) The Anopheles superpictus com- plex: introduction of a new malaria vec- tor complex in Iran. Bull Soc Pathol Exot. 101: 429–434. 4. Harbach RE (2013) the Phylogeny and Classification of Anopheles. In: Manguin S (Ed) Anopheles Mosquitoes-New In- sights into Malaria Vectors. InTech, Rijeka, Croatia, pp. 1–55. 5. Djadid ND, Jazayeri H, Gholizadeh S, Rad ShP, Zakeri S (2009) First record of a new member of Anopheles Hyrca- nus Group from Iran: molecular identi- fication, diagnosis, phylogeny, status of kdr resistance and Plasmodium infec- tion. J Med Entomol. 46: 1084–1093. 6. Doosti S, Yaghoobi-Ershadi MR, Schaffner F, Moosa-Kazemi SH, Akbarzadeh K, http://mosquito-taxonomicinventory./ http://mosquito-taxonomicinventory./ J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 249 Gooya MM, Vatandoost H, Shirzadi MR, Mostafavi E (2016) Mosquito surveil- lance and the first record of the invasive mosquito species Aedes (Stegomyia) al- bopictus (Skuse) (Diptera: Culicidae) in southern Iran. Iranian Journal of Public Health. 45: 1064–1073. 7. Azari-Hamidian S, Norouzi B, Noorallahi A (2017) Orthopodomyia pulcripalpis (Diptera: Culicidae), a genus and species new to the Iranian mosquito fauna, with a review of bionomical information. Zootaxa. 4299 (1): 141–145. 8. Gutsevich AV (1943) On the mosquitoes of north Iran. Compt Rend Acad Sci. 40(3): 123–125 9. Dow RP (1953) Notes on Iranian mosqui- toes. Am J Trop Med Hyg. 2: 683–695. 10. Sedaghat MM, Harbach R (2005) An annotated checklist of the Anopheles mosquitoes (Diptera: culicidae) in Iran. J Vector Ecol. 30(2): 272–276. 11. Hanafi-Bojd AA, Azari-Hamidian S, Vatandoost H, Charrahy Z (2011) Spa- tio-temporal distribution of malaria vec- tors (Diptera: Culicidae) across differ- ent climatic zones of Iran. Asian Pac J Trop Med. 4(6): 498–504. 12. Sofizadeh A, Moosa-Kazemi SH, Dehghan H (2017) Larval habitats characteristics of mosquitoes (Diptera: Culicidae) in north-east of Iran. J Arthropod-Borne Dis. 11(2): 211–225. 13. Sofizadeh A, Edalat H, Abai MR, Hanafi- Bojd AA (2016) Fauna and some bio- logical characteristics of Anopheles mos- quitoes (Diptera: Culicidae) in Kalaleh County, Golestan Province, northeast of Iran. Asian Pac J Trop Biomed. 6(9): 730–734. 14. Foster WA, Walker ED (2009) Mosqui- toes (Culicidae). Mullen GR, Durden LA. Medical and Veterinary Entomol- ogy. Elsevir, Oxford, pp. 207–260. 15. Edrisian GhH (2006) Malaria in Iran. Past and Present Situation. Iran J Parasitol. 1(1): 1–14. 16. Chinikar S, Shah-Hosseini N, Mostafavi E, Moradi M, Khakifirouz S, Jalali T, Goya MM, Shirzadi MR, Zainali M, Fooks AR (2013) Seroprevalence of West Nile Virus in Iran. Vector-Borne Zoonotic Dis. 13(8): 586–589. 17. Bahadori R, Eslami A (2007) Prevalence of blood filaria in dogs in Golestan Province (North of Iran) using Modified Knott Method and determination of its periodicity. J Vet Res. 62(1): 11–14. 18. Shahhosseini N, Chinikar S, Mossa-Kazemi SH, Sedaghat MM, Kayedi MH, Luh- ken R, Schmidt-Chanasit J (2017) West Nile Virus lineage-2 in Culex specimens from Iran. Trop Med Int Health. 22 (10): 1343–1349. 19. Azari-Hamidian S, Yaghoobi-Ershadi MR, Javadian E, Abai MR, Mobedi I, Lin- ton YM, Harbach RE (2009) Distribu- tion and ecology of mosquitoes in a fo- cus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes. Med Vet Entomol. 23: 111–121. 20. Azari-Hamidian S (2005) Larval habitat characteristics of mosquitoes of the ge- nus Culiseta Felt, 1904 (Diptera: Cu- licidae) in the Caspian Sea littoral, Iran. Zool Middle East. 36(1): 59–66. 21. Azari-Hamidian S (2007) Larval habitat characteristics of mosquitoes of the ge- nus Culex (Diptera: Culicidae) in Gui- lan Province, Iran. Iran J Arthropod- Borne Dis. 1(1): 9–20. 22. Azari-Hamidian S (2011) Larval habitat characteristics of the genus Anopheles (Diptera: Culicidae) and a checklist of mosquitoes in Guilan Province, north- ern Iran. Iran J Arthropod-Borne Dis. 5 (1): 37–53. 23. Azari-Hamidian S, Abai MR, Arzamani K, Bakhshi H, Karami H, Ladonni H, Harbach RE (2011) Mosquitoes (Dip- tera: Culicidae) of North Khorasan Prov- https://www.ncbi.nlm.nih.gov/pubmed/?term=Goya%20MM%5BAuthor%5D&cauthor=true&cauthor_uid=23697768 https://www.ncbi.nlm.nih.gov/pubmed/?term=Shirzadi%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=23697768 https://www.ncbi.nlm.nih.gov/pubmed/?term=Zainali%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23697768 https://www.ncbi.nlm.nih.gov/pubmed/?term=Fooks%20AR%5BAuthor%5D&cauthor=true&cauthor_uid=23697768 J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 250 ince, northeastern Iran and the zoogeo- graphic affinities of the Iranian and middle Asian mosquito fauna. J Ento- mol. 8(3): 204–217. 24. Nikookar SH, Moosa-Kazemi SH, Yaghoobi-Ershadi MR, Vatandoost H, Oshaghi MA, Ataei A, Anjamrooz M (2015) Fauna and larval habitat charac- teristics of mosquitoes in Neka County, northern Iran. J Arthropod-Borne Dis. 9(2): 253–266. 25. Nikookar SH, Azari-Hamidian S, Fazeli- Dinan M, Mousavi Nasab SN, Arabi M, Ziapour SP, Enayati A (2016) Spe- cies composition, co-occurrence, asso- ciation and affinity indices of mosquito larvae (Diptera: Culicidae) in Mazanda- ran Province, northern Iran. Acta Trop. 157: 20–29. 26. Nikookar SH, Fazeli-Dinan M, Azari- Hamidian S, Mousavinasab SN, Arabi M, Ziapour SPو Shojaee J, Enayati A (2017) Species composition and abun- dance of mosquito larvae in relation with their habitat characteristics in Ma- zandaran Province, northern Iran. Bull Entomol Res. 107(5): 598–610. 27. Abai MR, Saghafipour A, Ladonni H, Jesri N, Omidi S, Azari-Hamidian S (2016) Physicochemical characteristics of larval habitat waters of mosquitoes (Diptera: Culicidae) in Qom Province, central Iran. J Arthropod-Borne Dis. 10(1): 65–77. 28. Hanafi-Bojd AA, Soleimani-Ahmadi M, Doosti S, Azari-Hamidian S (2017) Lar- val habitats, affinity and diversity indi- ces of Culicinae (Diptera: Culicidae) in southern Iran. Inter J Mosq Res. 4(2): 27–38. 29. Golfiruzi S, Kourdi KH, Abolhasani M (2012) Statistics Yearbook of Health center in Golestan Province (2010–2011). Vol. 1. Noroozi Press, Gorgan, Iran (Per- sian). 30. Azari-Hamidian S, Harbach RE (2009) Keys to the adult females and fourth- instar larvae of the mosquitoes of Iran (Diptera: Culicidae). Zootaxa. 2078: 1– 33. 31. Lenhart AE, Walle M, Cedillo H, Kroeger A (2005) Building a better ovitrap for detecting Aedes aegypti oviposition. Ac- ta Trop. 96(1): 56–59. 32. Dibo MR, Chiaravolloti-Neto F, Battigaglia M, Mondini A, Favaro EA, Barbosa AAC, Glasser CM (2005) identifica- tion of the best ovitrap installation sites for gravid Aedes (Stegomyia) aegypti in residences in Mirassol, state of Sao Paulo, Brazil. Mem Inst Oswaldo Cruz. 100(4): 339–343. 33. Azari-Hamidian S, Joefshani MA, Moss- lem M (2005) Mosquitoes fauna and distribution of the genus Culex (Diptera: Culicidae) in Guilan Province. Pajouhesh and Sazandegh. 68: 39–45. (Persian). 34. Mousakazemi S, Zaim M, Zahraii A (2000) Fauna and ecology of Culicidae of the Zarrin-Shahr and Mobarakeh area in Isfahan Province. Armaghan Danesh, J Yasuj Univ Med Sci. 5: 46–54 (Persian). 35. Zaim M (1987) the distribution and lar- val habitat characteristics of Iranian Culicinae. J Am Mosq Control. Assoc. 3(4): 568–573. 36. Banafshi O, Abai MR, Ladonni H, Bakh- shi H, Karami H, Azari-Hamidian S (2013) The fauna and ecology of mos- quito larvae (Diptera: Culicidae) in west- ern Iran. Turk J Zool. 37: 298–307. 37. Ladonni H, Azari-Hamidian S, Alizadeh M, Abai MR, Bakhshi H (2015) The fau- na, habitats, and affinity indices of mos- quito larvae (Diptera: Culicidae) in Cen- tral Iran. N West J Zool. 11(1): 76–85. 38. Saghafipour A, Abai MR, Farzinnia B, Nafar R, Ladonni H, Azari-Hamidian S (2012) Mosquito (Diptera: Culicidae) Fauna of Qom Province, Iran. J Ar- thropod-Borne Dis. 6(1): 54–61. 39. Dehghan H, Moosa-Kazemi S H, Zahirnia J Arthropod-Borne Dis, September 2018, 12(3): 240–251 A Sofizadeh et al.: Fauna and Larval … 251 A, Davari B, Sharifi F (2011a) Larval habitat diversity and species composi- tion of mosquitoes (Diptera: Culicidae) in Hamadan Province. Sci J Hamadan Univ Med Sci. 18 (3): 50–58. 40. Dehghan H, Sadraei J, Moosa-Kazemi SH, Davari B (2011b) Ecological study of Culex pipiens, the dominant species of Culicidae mosquitoes and its control sou- lations in Yazd Province. Sci J Kurdi- stan Uni Med Sci. 15(4): 81–88 (Persian). 41. Moosa Kazemi SH, Karimian F, Davari B (2010) Culicinae mosquitoes in Sanan- daj County, Kurdistan Province, eastern Iran. Vector Borne Dis. 47: 103–107. 42. Khoobdel M, Azari-Hamidian S, Hanafi- Bojd AA (2012) Mosquito fauna (Dip- tera: Culicidae) of the Iranian islands in the Persian Gulf II. Greater Tonb, Lesser Tonb and Kish Islands. J Nat Host. 46 (31–32): 1939–1945. 43. Azari-Hamidian S, Linton YM, Abai MR, Ladonni H, Oshaghi MA, Hanafi-Bojd AA, Moosa-Kazemi SH, Shabkhiz H, Pakari A, Harbach, RE (2010) Mosqui- to (Diptera: Culicidae) fauna of the Ira- nian islands in the Persian Gulf. J Nat Host. 44(15): 913–925.