Mosquitoes belong to the Diptera order, Nematocera suborder, and Culicidae family. The Culicidae family is among the largest and most important families in the Nematocera suborder. Their bites have the potential to induce skin allergies in susceptible individuals, especially children, resulting in localized discomfort, secondary skin infections, and general unease ( Abai et al., 2007). Furthermore, mosquito bites have the capacity to disturb rest, triggering mental discomfort and nervous irritations ( Wilkerson et al., 2021, Berlov and Kuberskaya, 2023).

They are known as one of the most significant groups of arthropods concerning human health, as they serve as vectors for various pathogenic agents such as viruses, bacteria, protozoa, and nematodes. Mosquitoes are capable of transmitting numerous pathogens, including malaria, dengue fever, yellow fever, chikungunya, encephalitis, and filariasis.

There are more than 3,500 species of mosquitoes categorized into 112 genera globally. The Culicidae family is traditionally divided into two subfamilies: Anophelinae and Culicinae. Anophelinae includes about 500 species, mostly within the Anopheles genus, while Culicinae includes over 3,500 species across its 110 genera ( Cannet et al., 2023, Santos et al., 2017). The Anopheles, Culex, along with the Aedes, are significant genera of mosquito worldwide ( Knols, 2021). Out of 3578 mosquito species, 88 are known to transmit 78 different pathogens that cause diseases in humans. An additional 243 species are also identified as potential or likely vectors, making a total of 331 species that are associated with human diseases. ( Yee et al., 2022).

In southwestern Asia, there are seven genera and over 98 species of mosquitoes belonging to the Culicidae family. The most recent checklist of Iranian mosquitoes includes species from both subfamilies, totaling 70 species representing 8 or 12 genera, depending on the classification of aedines ( Jaberhashemi et al., 2022, Azari-Hamidian et al., 2019). In Iran, the transmission of West Nile virus by mosquitoes has been documented in various regions of the country, including the western areas of the Hyrcanian forests (Gilan province). Previous reports have also mentioned the possible transmission of Japanese encephalitis ( Azari Hamidian et al., 2011) and dengue fever ( Heydarifard et al., 2024) in Iran. Additionally, cases of Dirofilaria Immitis and D. repens Railliet transmission by these mosquitoes have been reported within the country ( Kartman, 1953, Pampiglione and Rivasi, 1997, Azari-Hamidian et al., 2009).

In southeastern Iran, the primary malaria vectors are Anopheles fluviatilis, An. dthali, An. stephensi, An. culicifacies, and An. superpictus. While Anopheles hyrcanus is not a significant malaria vector in Iran, a related species is noted in Afghanistan, where An. hyrcanus serves as the vector instead of An. dthali ( Moin-Vaziri et al., 2022).

There is limited data on the mosquito species present in the eastern regions of the Hyrcanian forests, specifically in Golestan Province. To date, 10 species of Anopheles and 14 species of Culicinae have been identified through their morphological traits and egg surface patterns. Earlier studies have primarily concentrated on other areas in northeastern Iran, such as Mazandaran and North Khorasan Provinces ( Sofizadeh et al., 2017).

Studying mosquitoes in a systematic way is crucial for understanding their biology and effectively managing diseases they transmit. The taxonomy of Culicidae has been a focus of attention due to their role in spreading diseases, making it an important area of research for medical and veterinary purposes ( Foster and Walker, 2019). Investigating the fauna and diversity of species allows for studying their distribution in the environment and providing recommendations for controlling medically significant species.

Golestan province is home to 8 of the 13 recognized climate zones worldwide ( Raziei, 2022). Our study aimed to examine the diversity, richness, and distribution of adult mosquitoes in the eastern regions of the Hyrcanian forests in northern Iran. Given the area’s significant species richness, we analyzed ecological indicators to assess the varying habitat conditions within the regions. This investigation enhances our understanding of mosquito populations and their ecological dynamics in this vital environment.

A total of 819 adults were collected from nine counties in Golestan Province, Iran between April and December of 2024. From the total collected samples, 491 female mosquitoes were morphologically identified using valid taxonomic key ( Azari-Hamidian and Harbach, 2009). In addition, 70 unidentified mosquitoes (due to loss of key identification characteristics during capture and handling), 23 male mosquitoes, and 235 non-biting midges were collected in this study.

We identified six species (four species of culicine mosquitoes and two species of anopheline mosquitoes) in all studied locations ( Table 2). The most abundant species (eudominat species) of mosquito collected was Cx. Tritaeniorhynchus Giles, 1901, which made up 84.93% of the total number of mosquitoes collected. An. hyrcanus Pallas, 1771 was the second most abundant species (subdominant species), making up 8.55% of the total number of collected mosquitoes. Cx. Theileri Theobald, 190 (1.02%), An. maculipennis Meigen, 1818 (1.83%), and Cx. Pipiens Linnaeus, 1758 (3.46%) were recedent species and made up a smaller percentage of the total number of mosquitoes collected. Furthermore, Cx. territans Walker, 1856 was a subrecedent species with the frequency of 0.20 % of all specimens ( Table 2).

Individual distribution maps and frequencies of all collected mosquito species, including An. hyrcanus ( Figure 2), An. maculipennis ( Figure 3), Cx. tritaeniorhynchus ( Figure 4), Cx. territans ( Figure 5), Cx. pipiens ( Figure 6), and Cx. theileri ( Figure 7), were prepared across Golestan Province using ArcGIS software.

The highest species diversity was observed in Galikesh, which had 5 of the 6 collected species, while Turkman recorded 4 species.

The highest geographical distribution in the study area was associated with Cx. tritaeniorhynchus, being found in all locations. In second place, Cx. pipiens was present in 6 out of the 9 regions studied. The lowest distribution was attributed to Cx. territans, which was only identified in the Galikesh region.

The estimation of species richness (S) and all main calculated diversity indices of adult mosquitoes in nine counties of Golestan Province, northeastern Iran are shown in detail in Table 3. The Shannon-Wiener Diversity Index for the mosquito community in this study was 0.5981. This index is a measure of the diversity of a community, taking into account both the number of species present and their relative abundance ( Table 3). The Simpson Dominance Index for the mosquito community in this study was 0.73. This index measures the dominance of the most abundant species in a community ( Table 3).

Based on the result of the pairwise similarity test for the mosquito fauna of the nine counties, the average pairwise similarity was calculated 0.593. Bandar Gaz – Turkman (3 common species), Gonbade-Kavus-Kordkuy (one common species), Gonbade-Kavus-Maraveh Tappeh (one common species), and Kordkuy-Maraveh Tappeh (one common species) displayed the most similarity (1) and Galikesh- Bandar Gaz (3 common species) placed in the second place with the similarity of 0.841. Moreover, Galikesh - Gonbade-Kavus, Galikesh-Kordkuy, and Galikesh - Maraveh Tappeh showed the least similarity (0.308) with one common species ( Table 4).

In this study, out of 491 adult mosquitoes identified, two species from the Anopheline subfamily ( An. maculipennis, and An. hyrcanus) and four from the Culicine subfamily ( Cx. tritaeniorhynchus, Cx. pipiens, Cx. theileri, and Cx. territans) were found. In this study, no mosquitoes of the genus Aedes were collected due to our sampling methods, which focused solely on adult mosquitoes. Additionally, other techniques, such as larval collection and ovitrap installation, were not used, representing another limitation of our research.

Almost all species captured in this study are of medical significance and may serve as potential disease vectors for humans and animals ( Soltan-Alinejad et al., 2021). The mosquito species An. maculipennis and An. hyrcanus are related to the transmission of malaria, a significant mosquito-borne disease. On the other hand, Cx. tritaeniorhynchus, Cx. pipiens, Cx. theileri, and Cx. territans are associated with the transmission of Dirofilaria immitis, a parasitic worm that causes heartworm disease in animals ( Azari-Hamidian et al., 2019).

In this study, no new species were reported; all species had already been documented in various regions of Iran by other researchers. The key focus is on the species composition and dominant species in each region, which vary significantly and will be discussed next.

Cx. tritaeniorhynchus is a vector of Japanese encephalitis virus (JEV), which is a significant mosquito-borne disease affecting feral pigs, native mammals, and humans. This mosquito species plays a crucial role in the transmission of Japanese encephalitis, a viral disease that can lead to encephalitis in humans and other animals ( Lessard et al., 2021, Longbottom et al., 2017).

This medical important mosquito species was reported in various cities and provinces of Iran including Yazd City (Golestan Province, Central Iran), Kalaleh County (Golestan Province), Mazandaran Province, and Guilan Province ( Shoraka et al., 2020, Tong et al., 2023, Sofizadeh et al., 2017, Azari, 2007).

Cx. pipiens is a vector of various diseases, including West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Rift Valley fever virus (RVFV), Sindbis virus (SINV), and filarial nematodes that cause filariasis in different parts of the world ( Farajollahi et al., 2011, Brugman et al., 2018). This species was reported in several cities across Iran. The cities where Cx. pipiens was identified include: Tehran (the capital city of Iran), Yazd City in central Iran, Hamadan, Neka (Mazandaran Province), and Mashhad (Khorasan-Razavi Province) in northeast of the country ( Karami et al., 2016, Azari, 2007, Salim-Abadi et al., 2016, Dehghan et al., 2013).

Cx. theileri is a recognized vector of pathogens responsible for dirofilariasis, Sindbis fever, and West Nile fever in Iran and adjacent regions ( Azari-Hamidian and Omrani, 2022). Cx. theileri is prevalent in the Afrotropical, Palearctic, and Oriental Regions, with a primary presence in the southern Palearctic region. In Iran, this mosquito exhibits a broad distribution, spanning across 28 out of the 31 provinces, establishing itself as one of the most plentiful species in the country ( Azari-Hamidian and Omrani, 2022, Moosa-Kazemi et al., 2021, Sharifi et al., 2022, Kayedi et al., 2020).

Cx. territans is not known to be a vector of any specific diseases to human and is considered a frog-feeding mosquito. This species is known to transmit Batrachochytrium dendrobatidis (Bd), a pathogenic fungus that affects amphibians, particularly frogs. This mosquito species can carry the fungus and potentially transmit it to amphibian hosts, contributing to the spread of Bd and impacting susceptible amphibian populations worldwide ( Reinhold et al., 2023). This mosquito was reported from Tehran, and Guilan Provinces in Iran ( Azari, 2007). Cx. territans predominantly inhabits natural swamps, ponds, and pools.

An. maculipennis and An. hyrcanus are vector of malaria, a significant mosquito-borne disease. These species are known to transmit Plasmodium parasites, which cause malaria in humans and other animals. Malaria is a major public health concern worldwide, with an estimated 229 million cases and 409,000 deaths in 2019 ( Fikadu and Ashenafi, 2023). These two main vectors were reported in some northern regions of Iran including Ramsar, Sari, Babolsar, and Chalous (Mazandran Province), and also Astaneh on the border of Guilan and Mazandran Provinces ( Djadid et al., 2007, Azari-Hamidian et al., 2006).

In Qom Province, an investigation was carried out on a collection of larvae mosquitoes, revealing 14 species (4 Anophelinae and 10 Culicinae). Cx. hortensis, Cx. theileri were reported as dominant species in their study. Also, Cx. pipiens, Cx. territans, and Cx. theileri were the catch species similar to our study ( Saghafipour et al., 2012).

Similarly, in Kurdistan Province, a comprehensive study was conducted to identify the mosquito species, which resulted in the collection of four genera and 11 species. An. superpictus was the most abundant and widely distributed species. Furthermore, An. maculipennis, Cx. pipiens, and Cx. theileri were similar reported species to the results of the present study ( Banafshi et al., 2013).

Seven genera and 15 species of mosquitoes were identified in East Azarbaijan Province. An. hyrcanus, An. maculipennis s.l., Cx. pipiens, Cx. theileri, and Cx. tritaeniorhynchus were common species with our study. In East Azarbaijan Province, An. sacharovi was dominant species during all collected specimens and Cx. theileri was also the most dominant species between culicinae species ( Abai et al., 2007).

In Khuzestan Province, several species of culicid mosquitoes are recorded, including An. stephensi, An. pulcherrimus, Aedes caspius, Cx. tritaeniorhynchus, Cx. quinquefasciatus, and Cx. theileri. Similar to the results obtained in our research, Cx. tritaeniorhynchus was the dominant species identified in Khuzestan, and also two species similar to our study were reported in this study ( Faraji-Fard et al., 2021).

Recently 16 culicine species were reported in Hormozgan Province. In that study among adults, Cx. laticinctus was the most prevalent species ( Jaberhashemi et al., 2022).

Nine species of mosquitoes were identified in Neka, Mazandaran Province. Cx. pipiens with the frequency of 63.99% was the eudominant species among all collected mosquitoes. Although Mazandaran Province neighbors Golestan, the dominant species in that region differs from our study ( Nikookar et al., 2015).

In two Provinces (Kurdistan and Kermanshah) in the west of Iran, five genera and eleven species of mosquitoes were identified. Culiseta longiareolata and Cs. subochrea were reported as predominant species in larval collection, and Cx. theileri was reported as dominant in Adult stage ( Moosa-Kazemi et al., 2015).

Five genera and 12 species of mosquitoes were identified in a study conducted in West Azerbaijan Province. Consistent with our own findings, An. maculipennis, Culex pipiens, and Cx. theileri were among the specimens collected in that region ( Khoshdel-Nezamiha et al., 2014).

In Firouzabad County, located in Fars Province, a total of 6 species of Culicidae were documented. Among these species, the only common species with our results was Cx. theileri. This species also was dominant among collected specimens at Firouzabad ( Soltani et al., 2017).

Three genera and eight species of mosquitoes were collected in Hamedan County, Hamedan Province. Like the results obtained in our research, Cx. theileri, Cx. pipiens, and An. maculipennis was reported. In Hamadan, Cx. theileri was the most prevalent species among all mosquitoes.

Three genera and eight species of mosquitoes were collected in Hamedan County, Hamedan Province. Consistent with our research findings, Cx. theileri, Cx. pipiens, and An. maculipennis were among the reported species. Notably, in Hamadan, Cx. theileri emerged as dominant species among all mosquitoes ( Zahirnia and Zendehfili, 2014).

A comprehensive faunal study of the mosquitoes was done in Mazandaran Province, Northern Iran. Totally, 4 genera and 20 species of mosquitoes were identified and Cx. pipiens was introduced as the eudominant species followed by Cx. tritaeniorhynchus and An. maculipennis ( Nikookar et al., 2018). While the dominant species of mosquitoes in Mazandaran differed from our research findings, Cx. tritaeniorhynchus emerged as the second dominant species, consistent with its prevalence in our study.

The biodiversity analysis conducted in this study revealed an evenness index (J’) of 33.4%, which indicates a clear disparity in the abundance of different species within the community. This low evenness suggests that the various species present do not share the available resources equally, highlighting an imbalance in their populations. Furthermore, a higher Simpson Dominance Index implies that one species is significantly more prevalent than the others in the sampled areas. Based on our findings, with a Simpson Dominance Index of 0.73, we can confidently conclude that Cx. tritaeniorhynchus has emerged as the predominant mosquito species in the areas we studied. This dominance points to a concerning trend of low overall species diversity among mosquitoes in the Hyrcanian forests. The consistent and stable weather and climate conditions prevalent in this region have created an environment that allows Cx. tritaeniorhynchus to thrive exceptionally well, enabling it to occupy and dominate most of the available ecological niches.

The distribution of mosquito species varies across different regions of Iran. Although the species identified in our study were not new in the country, the dominant species in the Golestan region differed significantly from other parts of the country. This variation can be attributed to various factors, including distinct sampling methods, the number of sampling locations, climatic variances, and other environmental influences. Moreover, the current study has recognized potential or confirmed vectors of pathogens affecting humans and domesticated animals in Golestan Province, underscoring the significance of exploring the bioecology of these vectors in unexplored regions in Hyrcanian forests.

It is important to note that this data is from a single study in a specific location and time period. Therefore, it cannot be used to draw general conclusions about mosquito diversity in other regions or over time. We created distribution maps for each mosquito species in the surveyed areas and identified the dominant species in each community. Conducting faunal studies on mosquitoes across various regions of Iran offers valuable information on the biodiversity and distribution of mosquito species. This data serves as a fundamental resource for implementing effective management strategies against mosquito-borne diseases. The distribution of mosquito species varies across the country, and there is a need for further studies to understand the ecology of these species and their potential impact on public health.

All study procedures were conducted in compliance with the Declaration of Helsinki and were approved by the Iran National Committee for Ethics in Biomedical Research (Approval ID: IR.SUMS.REC.1402.536) on 17 February, 2024. Written consent was obtained from all participants before the study commenced.