Biodiversity of Mosquitoes (Diptera: Culicidae) with Emphasis on Potential Arbovirus Vectors in East Azerbaijan Province, Northwestern Iran.

Background
The abundance, diversity, distribution and ecology of mosquitoes (Diptera: Culicidae), especially arbovirus vectors are important indices for arthropod-borne diseases control.


Methods
Larvae and adult mosquitoes were collected using the standard methods from different habitats in nine localities of three counties in the East Azerbaijan Province, Northwestern Iran during June to October 2017. In addition, species richness (R), Simpson's diversity index (D), Shannon-Wiener index (H') and evenness (E) as measures of diversity, were calculated.


Results
Overall, 1401 mosquito specimens including 1015 adults and 386 larvae were collected in the study area. The properties of geographical larval habitats were recorded. Four genera along with 10 species were collected and identified, including Anopheles hyrcanus, An. maculipennis s.l., An. superpictus s.l., Aedes caspius, Ae. vexans, Culex pipiens, Cx. theileri, Cx. perexiguus, Culiseta longiareolata and Cs. subochrea. Among the three counties, Ahar region presented the highest species richness (R: 1.5) and diversity values (D: 0.79, H': 1.74, E: 0.73).


Conclusion
This study provides important information on the diversity, distribution and ecology of ten mosquito species in the region. This information leads to a better understanding of mosquito population dynamics in relation to vector control measures.


Introduction
Mosquitoes (Diptera: Culicidae) are suborder of Nematocera and Culicidae family, which are the medically-important species for malaria transmission, types of filariasis, types of encephalitis and other arboviral diseases (1)(2). The Culicidae family includes 2 subfamilies, 11 tribes; The Culicidae family includes 2 subfamilies, 41 Families, 11 tribes, 113 genera and 3563 species (1)(2)(3)(4). The most important genera of the Culicidae family are Anopheles, Culex and Aedes. Among the Culicidae, the Aedes species have the most various species and are extremely important in medical cases (5).
Mosquitoes play a crucial role in transmission of some important diseases such as malaria, filariasis, dengue fever, yellow fever, chikungunya, West Nile virus and Zika virus which are today among the greatest health problems in the world (6-9).
Mosquitoes larvae have the power to col-onize and live in a wide variety of natural and artificial habitats, such as temporary and permanent water resources, unclean and clean water, large or small water resources, stagnant or stream waters and even the smallest places where water is gathered, such as buckets of water, pots, tires, bromeliads, animal feet prints and plant leaf axes (10)(11). Adult mosquitoes are very diverse bionomically, for example, they are too diverse in host searching, biting behaviors, dispersal and reproductive strategies (9). Two factors (abiotic and biotic) affect mosquito life cycle. The biotic factors contain adult sugar and blood meal types, species communications, interactions and natural enemies. Physicochemical attributes of larval habitats are abiotic factors, which include the type and contents of water, temperature and rainfall.
There are complex interactions between these factors that significantly affect mosquito ecological adaptability and vectorial capacity for disease transmission with significant concepts for vector management and control at the local and regional levels (12)(13). Accordingly, investigating biotic and abiotic factors for various mosquito fauna make it easier to monitor potential modifications of larval habitats affected by rains, global climate change and man-made activities (12)(13).
Aedes caspius likely transmit the human pathogen, such as Spiroplasma sabaudiensc and Crisiulospora aedis (14). These mosquitoes transmit Rift Valley fever virus (RVF), West Nile virus (WNV) and Tahyan virus (15). In the region of Palearctic, Ae. caspius inhabits in lakes, pools, shores of Great Britain, and fresh-water and lower salt marshes in the continental parts of Europe, Russia, Mongolia, northern China, Pakistan, northeastern Africa, Asia Minor, and the Persian Gulf (17)(18).
Aedes vexans has several subspecies, the typical species is Ae. vexans vexans, and the subspecies Ae. nipponii vexans in East Asia (19) and Ae. vexans arabiensis in Saudi Arabia, Yemen and Africa (3). This species is extremely important in medicine and has more than 30 different viral diseases, including Eastern horse encephalitis, Japanese encephalitis (20), California encephalitis, Western horse encephalitis (21), as well as other pathogens such as Tularemia and Dirofilaria immitis (22)(23). Aedes vexans along with Ae. aegypti and Cx. quinquefasciatus, have the highest global distribution among other mosquitoes in the world (24)(25).
Several studies were conducted on composition, distribution and ecology of mosquitoes in Iran (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). Considering the fact that in the East Azarbaijan Province, some vector mosquitoes of diseases such as West Nile, Dirofilariasis and malaria present, study on the composition, distribution and ecology of mosquito species is very important in different aspects of vector control programs. This study will add important information about the composition, distribution, ecology and diversity of mosquito species such as establishment of mosquitoes in this region. This new and important information will help us to correctly control and monitor strategies of disease vectors, such as Ae. caspius, Ae. vexans, Cx. pipiens and Cx. theileri. This information helps us to prevent important vectors from increasing and establishment in this area due to change in human activities and weather; thus, the risk of transmission of disease by mosquitoes gets minimized.
The aim of this study was to determine composition, distribution and some ecological aspects of mosquitoes in East Azerbaijan Province, Iran which is high medically importance from the point of view of the arboviral vectors.

Study area
East Azerbaijan Province is located in northwestern of Iran between 39º 26´-36º 45´ N latitudes and 45º 5´-48º 22´ E longitudes. The province covers an area of approximate-  Table 1).
This province contains 19 counties. Tabriz City is center of the province which 1360m above sea level. The mean annual rainfall is about 300mm. The average relative humidity changes are from 44%. The averages of the maximum and minimum temperatures are 17.7 °C and 6.8 °C, respectively, and the average temperature is 12.3 °C. The province includes arid and semiarid climates (Fig. 1, Table 1).

Mosquito collection, site selection and species identification
For mosquito collection, nine sites with different biotopes in three counties of Ahar, Marand, and Tabriz from East Azerbaijan Province were selected.
Collection of mosquito's larvae was performed in different habitats using the standard dipping technique (using 350ml Clark's dippers) and whirl pack bags (45) ( Table 2). Light trap, mosquito net and aspirator was used for adult mosquito collection. Collection of mosquitoes was carried out during June to October 2017. Larvae and adult mosquitoes were identified by morphological characters (46).

Physical and biological characteristics of larval habitats
In these investigation characteristics of mosquitoes larval habitats was studied. The ecological characteristics containing geographical properties of collecting localities (latitude, longitude and altitude), type of habitat (stagnant, stream, seepage and water container), vegetation situation (presence or lack of vegetation), kind of vegetation (Null, Poaceae, Typhaceae, Dispacea and Acorus species), water situation (turbid and clear), exposure to sunlight (full, partial sunlight and covered or shaded), depth, substrate type (muddy, sandy, rocky and concrete), distance from animal and human houses and physicochemical attributes such as water temperature and pH were recorded visually or using special equipment (9,47).

Data analysis
The species richness (R: Margalef index), unified indices (D: Simpson's diversity index and H ̕ : Shannon-Wiener index) and evenness (E: distribution of abundances among the species) as measures of a diversity, were calculated for East Azerbaijan Province and different study counties.
The formulae and their rationale in the present study are summarized below:

Results
Totally, 1401 mosquitoes were collected from nine sites in East Azerbaijan Province during June-October 2017, including 1015 adult and 386 larvae. The properties of geographical larval habitats (Latitude, Longitude and altitude) have been shown in (Fig. 1 Cs. longiareolata (Table 2) are new report for the region.
Species such as Cx. pipiens, Cx. theileri, Cs. longiareolata and Ae. vexans have a wide distribution in the study areas but some species such as An. hyrcanus, An. maculipennis s.l., An. Superpictus s.l., and Cs. subochrea have been collected from limited areas (Table 2).

Characteristics of mosquito larval habitats of mosquitoes
In Ahar County located in northeastern East Azerbaijan Province, there were three larval sites such as Yavarkandi, Razin and Sattar Khan Dam (Table 3, Fig. 1). Sattar Khan Dam and Razin larval sites had seepage and stagnant water respectively. Both of the larval sites had turbid water with muddy substrate and shallow depth. In addition, these sites were covered from sunlight with Null, Poaceae, Typhaceae and Dispacea vegetation. Those were more than two kilometers away from human and animal houses. The Sattar Khan Dam larval habitat had 200 meters distance from animal space and its field temperature was 13 °C. In contrast, Razin larval site had 500 meters distance from animal space and its field temperature was 9 °C. In these two larval habitats harbored Ae. caspius, Ae. vexans, Cx. theileri, Cx. pipiens, Cx. perexiguus, Cs. subochrea and An. superpictus s.l. (Table 2). Yavarkandi larval site had stagnant and clear water without vegetation, with muddy substrate and exposed to sunlight. It was more than two km away from human and animal houses. In the Yavarkandi larval site the field temperature was 23 °C. In this larval habitat, two species of Cs. longiareolata and Cx. pipiens were collected (Table 3).
Tabriz larval site is located in the central East Azerbaijan Province and contains three regions of Hojjaj Park, Khaje-dizaj and Chavan (Table 3, Fig. 1). Both regions of Hojjaj Park and Khaje-dizaj had stagnant and turbid water with muddy substrate. The Hojjaj Park had the following conditions: the parts of larval habitat were covered with Poaceae plants and its water depth was shallow, also it had more than two kilometers distance from animal and human houses. The field temperature was 17 °C in this site (Table 3). This site contains Ae. caspius, Cx. theileri, Cx. pipiens, Cx. perexiguus and Cs. longiareolata ( Table 2). The Khaje-dizaj site was covered with Null and Ziziphu plants. Its depth was less than one meter and its field temperature was 16 °C. It had one-kilometer distance from human houses and about 100 meters from animal sites (Table 3). In this larval habitat, Cx. theileri and Cx. pipiens were collected (Table 2). Chavan larval habitat was a water container with substrate cement and clear water that had no plants and was exposed to sunlight. It had a very short distance with human and animal houses (About 2-10m), which had one meter depth and 27 °C (Table 3). In this larval habitat Cs. longiareolata and Cx. pipiens were collected ( In Marand with three larval sites of Ghareh Tappeh, Dolat Abad and Zinab located in northwest East Azerbaijan Province (Fig. 1). Ghareh Tappeh and Dolat Abad habitats had stagnant and clear water. Ghareh Tappeh site had no plants with sandy substrate and shallow depth that was exposed to sunlight. It had one meter depth and 18 °C field temperature (Table 3). Culiseta longiareolata, Cx. pipiens, Ae. vexans, An. maculipennis s.l. and An. hyrcanus were collected in this larval habitat ( Table 2). Dolat Abad site was shallow with muddy substrate and 18 °C field temperature. The parts of larval habitat were covered with Null and Dispacea plants and it had more than two kilometers distance from human and animal houses ( Table  3). In this larval habitat Cx. theileri and Cx. pipiens were collected ( Table 2). Zinab site was stream and clear water with rocky substrate and shallow depth. It had no plants and exposed to sunlight with 17 °C temperature. It had about 100 meters distance from human and animal houses (Table 3). In this larval habitat, specimens of Cx. pipiens, Cs. longiareolata, An. maculipennis s.l. and An. hyrcanus were collected (Table 2, Fig. 1).
According to Table 2 and 3, larvae of mosquitoes occupied all different types of the habitats. In addition, our results showed that Cx. pipiens, Cs. longiareolata and Cx. theileri, respectively, had the most distribution and ad-aptation to different types of larval habitats. These three species were collected in most larval habitats but in contrast, Cx. perexiguus was found only in the shallow water that was stagnant, turbid, covered with plants and with muddy substrate. In addition, distribution of Cs. subochrea was limited and only was collected in the seepage, stagnant and turbid water habitats with muddy substrate and shallow depth. Anopheles was found only in stream, stagnant and clean water with muddy substrate that was exposed to sunlight. Aedes vexans and Ae. caspius were found mostly in larval habitats with turbid water and they were covered with different types of plants. Only two Ae. vexans specimens were found in clean water that was exposed to sunlight with muddy substrate ( Table 2, 3). In our study, the temperature and pH ranges were 9 °C to 27 °C and 7-8 respectively.
There were differences in the species diversity, as indicated by the values of Simpson's diversity index, Shannon-Wiener index (H'), and evenness and species richness of the mosquito fauna among the study areas of East Azerbaijan Province (Table 4). The species richness and the three indices were found to be maximal in Ahar County (R: 1.

Discussion
This research is the first study on distribution, diversity and ecology of mosquitoes, with emphasis on Ae. capius and A. vexans as potential arbovirus vectors in East Azerbaijan Province, northwestern of Iran. The East Azerbaijan Province contains diverse geographical areas with different weather conditions. These diverse conditions can provide suitable environment for the establishment of different species of mosquitoes and justify the variety of mosquito species in this region. This study showed many mosquito species had ecological adaptations. In spite of these ecobiological characteristics, the ecology of mosquitoes present in East Azerbaijan Province is largely unknown. In this investigation we tried to study distribution and ecology of mosquitoes in three northeastern, northwest and central regions of East Azerbaijan Province, where various mosquito vectors of malaria and arboviruses are present (48)(49). Some studies had been conducted on fauna and checklist of mosquitoes in parts of this region (48), although to our knowledge no studies have been done on the ecology of mosquitoes in these regions.
In current study, 4 genera and 10 species were collected and identified. For the first time, Ae. caspius, Ae. vexans, Cx. perexiguus, Cs. subochrea species were reported from Ahar county. Also for the first time, Ae. vexans and Cx. perexiguus species were reported from Tabriz county. As well as for the first time, An. hyrcanus, An. maculipennis s.l., Ae. vexans, Cx. pipiens, Cx. theileri and Cs. longiareolata species were reported from Marand county. Some previous studies had found only An. hyrcanus in northwestern Iran (49)(50) and other study identified An. claviger, An. hyrcanus, An. maculipennis s.l., An. pseudopictus, An. sacharovi and An. Superpictus s.l. in East Azerbaijan Province. In the present study, we reported An. hyrcanus, An. maculipennis s.l. and An. Superpictus s.l.. In previous studies, Cx. pipiens and Cx. theileri were reported in these regions (48), but Cx. perexiguus was not found in these regions. In this study, three species of Culex genus were reported such as Cx. pipiens, Cx. theileri and Cx. perexiguus that Cx. pipiens and Cs. longiareolata species were the most abundant. Aedes caspius and Ae. vexans were found only from the Kaleybar region (49), In our study, for the first time, Ae. caspius and Ae. vexans were found in Ahar, Marand and Tabriz, where previous study did not report these species in these areas (48). One study reported Cs. annulata and Cs. subochrea in West Azerbaijan Province and Cs. subochrea in East Azerbaijan Province (51). Culiseta longiareolata species was reported as the most abundant in Kermanshah, Kurdistan and Sistan and Baluchistan provinces (52). In this study, two members of Culiseta genus were found, including Cs. longiareolata and Cs. subochrea where Cs. longiareolata species was the most abundant species.
Comparing the results of our study with a recent study in West Azerbaijan Province (47) showed that six species An. maculipennis s.l., An. superpictus s.l., Cx. pipiens, Cx. theileri, Cs. longiareolata and Ae. caspius were common between West and East Azerbaijan provinces. The results of our study compared with the results of research conducted in Zanjan Province (53), showed that five species (An. maculipennis s.l., An. superpictus s.l., Cx. pipiens, Cx. theileri, and Cs. longiareolata) were common between these two provinces. The comparison the results of our study with a recent study conducted in Kurdistan Province (54) showed that six species (An. maculipennis s.l., An. superpictus s.l., Cx. theileri, Cx. pipiens, Cs. longiareolata and Ae. caspius) were common between in provinces. In Turkey (55), and in the provinces of Ardebil, Kurdistan and West Azarbaijan, Cx. theileri and Cx. pipiens were dominant and most abundant species (56)  information. Our results showed that Cx. theileri (50.72%), Cs. longiareolata (32.12%) and Cx. pipiens (9.75%) were dominant and the most abundant species in this province.
The climate changes and biotic and abiotic environment factors including plants, temperature and rainfall ranges, significantly affect the type and frequency of larval mosquito habitats. These factors, in turn, affect the number of mosquito species, larval stages, longevity, behavior and adult development. As a result, the transmission of diseases through mosquitoes is directly affected by environmental factors (57)(58).
The presence of plants as a source of sugar for mosquitoes is very important, that influence both larval and adult stage development (59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70). Plants provide energy for mosquitoes, As a result, survival rate increased and longevity of the mosquito longer than the extrinsic incubation period of parasite, therefore the incidence of disease increases (61)(62). In our study, five types of plants such as Null, Poaceae, Typhaceae, Carex dipsacea and Acorus species were found in relation to mosquito larval habitats. To our knowledge, there is no study on plant species associated with mosquito species in Iran. In our study, various species of mosquitoes such as Anopheles, Culex, Aedes and Culisita were found in relation with various plants, therefore this study confirms the results of previous research. Aedes vexans and Ae. caspius have not been reported earlier in these areas. These results are likely to show the effects of climate changes and human activities on the distribution of these species in these regions.
Physicochemical properties of larval habitats regulate the abundance of mosquito species, for example, Anopheles species were found more in natural larval habitats and Culex species in artificial larval habitats (63). In our study, Anopheles species were found in natural habitats, while Culex species were collected from different types of larval habitats, indicating these mosquitoes can live in a wide range of water habitats. Larval habitats in this study were stagnant, stream and seepage, water container, turbid and clear water, sun exposed or covered from sunlight -rocky or muddy substrate and shallow depth. Studies showed that some of Culex larval species were found alone or along with other mosquitoes, such as Anophele and Aedes (64-67) which has been observed in our study.
There was a significant relationship between the distance of larval habitats of Anopheles mosquitoes, human and animal sites, besides, Anopheles mosquitoes are found more often near human and animal houses (68). Our study showed that there was a significant correlation between the distances of larval habitats of Anopheles, Ae. caspius, Ae. vexans and Cx. theileri mosquitoes with human and animal sites, because the larval habitats of these mosquitoes were found more near to human and animal houses. However, this correlation was not observed between the larval habitats of Cx. pipiens and Cs. longiareolata with human and animal houses, because the habitats of these mosquitoes were found at various intervals from human and animal houses.

Conclusion
Arbovirus vectors such as Ae. caspius and Ae. vexans along with Cx. pipiens and Cx. theileri are well adapted to a broad range of habitats and climatic conditions. Determining of distribution and full description of ecology of arboviral vectors under local eco-demographic conditions in the East Azerbaijan Province have provided important ecological information on establishment of important mosquito borne diseases. This new information will help us to correctly control and monitor strategies of disease vectors, and in this way prevent important vectors such as ae. Ae. Caspius, Ae. vexans, Cx. pipiens and Cx. theileri to be increased and established in this area due to changing human activities and weather chang-