Investigation on the Occurrence of Aedes Species in Borderline of Iran and Azerbaijan for Control of Arboviral Diseases.

Background
To investigate the diversity of the genus Aedes present in the natural areas of Ardabil Province, north-west of Iran.


Methods
This cross-sectional study was carried out from Apr to Oct 2016 in North-western of Iran. Thirty-three areas of 10 cities which are border areas were selected randomly. The larvae were collected 2 times in each month during the seasonal activities of mosquitoes and the larvae were identified morphologically according to the appropriate identification keys.


Results
Overall, 694 larvae were collected from four counties, from which only 7.2% were Aedes larvae. Three species of Aedes were identified which include Ae. caspius, Ae. vexans and Ae. flavescens. Aedes flavescens is reported from Ardabil Province for the first time.


Conclusion
Aedes species were a high density in borderline of Iran and Azerbaijan. Therefore, the north parts of Ardabil Province are a suitable habitat for Aedes species mosquitoes. Care should be taken for vector control in the case of occurrence of any arboviruses transmitted by Aedes mosquitoes.


Introduction
Mosquito-borne arboviruses are health threat in the worldwide. For instance, more than 2.5 billion people live in high-risk areas of dengue fever (DF) in the world and over 100 million people are infected with this disease (1,2).
Currently, there is no vaccine and no specific treatment for DF (3). Recently, outbreaks of DF have happened in Malaysia, Taiwan, and India (4). Endemic DF always occurred in Southeast Asia (5). However, some outbreaks recently have been occurred in parts of the Middle East, South Asian countries including Pakistan (6,7). Some factors affecting the distribution of DF in different parts of the world are as follows: Increasing urban population density, increased travel, and unsystematically urbanization (8)(9)(10). DF has been reported in 120 countries (11). The agent of DF is a Flaviviridae family and the main vectors are Aedes aegypti and Ae. albopictus (12). Thus mosquitoes are an invasive species in world widespread in tropical and temperate regions of the world. The ability to lay eggs and grow in dishes cultural artifacts. In the last two decades and facilitate the movement in the world are impacts factors in distribution of this mosquitoes (13).
Aedes aegypti and Ae. albopictus are vectors of important diseases such as DF, yellow fever and Chikungunya (14)(15)(16). Aedes aegypti is a mainly urban vector and is feeding exclusively from human (17). Aedes albopictus are mostly found in suburban and rural environments and are feeding of the different species of mammals, including humans, as well as the different species of birds (18).
Currently, vector control is the best method to control of the DF (19). In Iran, studies regarding the biodiversity and distribution of Aedes are limited. However, there is no information on Aedes mosquito's diversity components in north-western of Iran. The aim of this study was to investigate the diversity of Aedes genus present in the natural areas, as well as the differences on the faunestic composition of Aedes species in function of the climatic and ecological features of Ardabil Province borderline.
Various Aedes mosquito species are considered as potential

Study area
This cross-sectional study was carried out from Apr to Oct 2016 in north-western of Iran. Ardabil Province is located in northwestern Iran 37.45° to 39.42° N and 47.30° to 48.55° E. The province has an area of 17 953km 2 . This province is bordered to the north with the Republic of Azerbaijan and along the border is 282.5km (Fig. 1). In 159km from the border, flowing Aras and Balha rivers. During the border, Iran linked to the Republic of Azerbaijan for two areas Bilehsavar and Aslanduz. Ardabil Province in the longitudinal axis of the expansion (1°35`) and high extent to the north-south latitudes (2°31`) have a large va-riety of climates. About 2/3 textured mountainous with large variation in height and the rest are flat areas and posts. North province (Mugan plain) with low altitude has relatively warm weather and central and southern regions have a cool mountain climate (20)(21)(22).

Sample collection
Overall, 33 areas of 10 cities of priority border areas were selected randomly. During the seasonal activity, the larvae were collected in each month 2 times. Using a ladle handling, and the standard ladle of 350mL was collected from natural and artificial larval habitats. In each habitat, sampling was collected from different parts and the ladle was made 10 times on each side. In the case of well water used from the bucket and the limited larval habitats such as cavity trees were used from droppers. The larval stages III and were stored in lactophenol solution and after about a week and transparent larvae, using Berlese's Fluid were prepared microscopic slides and identified morphologically using appropriate identification keys.

Results
Overall, 2000 mosquito larvae were collected, from which only Aedes larvae were selected and identified. From 33 areas, six (18%) were positive for the presence of Aedes larvae. In four counties, (40%) Aedes larvae were collected. Totally, 694 larvae were collected from four counties that 7.2% were Aedes larvae. Three species of Aedes genus were identified which included Ae. caspius, Ae vexans, and Ae. flavescens. These species were reported from thee Ardabil Province for the first time. All three species were collected from 78-2114 meter above sea level altitude. Aedes larvae were collected from two different climate zones. The first zone: the northern part of the province where the climate is hot and humid and low altitude (60-78km) http://jad.tums.ac.ir Published Online: June 24, 2019 that includes Pars Abad and Bilehsavar and the second zone: southern part of the province with mountainous climate and high altitude (2114-2110m), which includes the Khalkhal and Sareyn (Table 1).
In the first zone the larvae were collected in Jun and Sep months but in the second zone in Jul and Aug. All three species Aedes more in border areas The maximum Aedes larvae from three species were collected in borderline of Iran and Azerbaijan but in other areas in this study, only two species for low abundance were collected ( Table 2). The survey of larvae habitat characteristics showed that most of larvae were collected (66%) in areas without trees and shrubs and sunny. Totally 80% of larvae habitats were temporary that including holes and marshes around rivers and craters were caused by the overflow waters and 86% were natural habitat. About 50 % of the larvae collected in the afternoon and at cooler temperatures and 33% at mid-day and 17% at AM. The water of larval habitat more was mainly stagnant and 50% transparent and type of bed habitat was 80% clay and 20% sand.

Discussion
In this study three spices of Aedes larvae (Ae. caspius, Ae. vexans and Ae. flavescens) identified from Ardabil Province, North-western of Iran that one species (Ae. flavescens) was reported for first time. Aedes larvae were dispersed in all regions of Ardabil Province but the frequency of Aedes larvae were maximum in north regions of this province that borderline of Iran and Azerbaijan. Only two adult species was reported (Ae. caspius and Ae. vexans) (22). Aedes caspius is distributed in Palearctic areas (23) and in Iran reported from more than 16 provinces such as Gilan, West Azerbaijan, Khorasan, Zanjan, Lorestan, Isfahan, Yazd, Kerman, Hormozgan, Bushire and Khozestan (24). This species very more frequently collected from six regions (Parsabad, Aslanduz, Bilehsavar, Khalkhal and Sareyn) in this study that two areas (Aslanduz and Bilehsavar) were located in borderline of Iran and Azerbaijan and both are customhouse. This species is also collected with extensive distribution from Gilan and Ardabil Province (25)(26). Moreover, Ae. caspius reported from Bushire (27), Eastern of Iran (28), Chaharmahal and Bakhtiari (29), East Azerbaijan (30) and Qom Province (31). In 2016, Ae. caspius along with Ae. albopictus and Aedes unilineatus reported from Sistan and Baluchestan (32). Aedes caspius more feeding on mammals and humans (9,33) and can be transmitted Rift Valley fever, dirofilariasis and tularemia (34).
Aedes vexans is distributed in Holoarctic and Oriental areas (35). This species and Ae. eagypti and Culex quinquefasciatus have the most distribution in world (33). In Iran also reported from Gillan, West Azerbaijan, Mazandaran, Bushire and Hormozgan (23). In this study Ae. vexans larvae were collected 2 times in Jun and Sep from Parsabad and Aslanduz. The number of this species were lower in comparison to other Aedes species but in Gillan Province the most larvae collected were Ae. vexans (25). The results of this study showed http://jad.tums.ac.ir Published Online: June 24, 2019 that the larvae of Ae. vexans were collected of less than 200m altitude that this result matched to another study (22) and the adults of this species collected from east Azerbaijan in high altitude (30). The feeding preference of this species is on the mammals then birds and reptiles (32). Aedes vexans can transmitted various diseases such as eastern and western equine encephalitis, Japanese encephalitis and California encephalitis.
Aedes flavescens was reported first time in Ardabil Province and 11 larvae of Ae. Flavescens collected from three parts of this area (Parsabad, Bilehsavar and Khalkhal). Totally 100% of larvae habitats were temporary and most whit vegetation. Total of these larvae were collected at afternoon. Zaim et al. for first time reported Ae. flavescens from West Azerbaijan in 1987 and one time from large pool whit vegetation (24). These results showed that Ae. flavescens were dispersed in northwest of Iran.

Conclusion
Aedes species were a high density in borderline of Iran and Azerbaijan. 40% of Ardabil Province was found Aedes mosquitoes. So the north parts of Ardabil Province are a suitable habitat for Aedes species mosquitoes. Therefore, more studies need to be done in these areas.