Mosquito (Diptera: Culicidae) diversity and medical importance in Koh Kong mangrove forests, Cambodia

In total 3107 mosquitoes were collected, of which 89.4% (2777 specimens) were identifiable to the species level (Table 1). During the study, 34 species from 10 genera were collected. The collection included 11 species of Culex, 5 Aedes, 4 Mansonia, 3 Coquilletidia, 3 Mimomyia, 3 Uranotaenia, 2 Anopheles, 1 Armigeres, 1 Heizmannia, and 1 species of Verralina.

The genus Culex, besides being the most diversified, was also the most abundant with 2571 specimens, representing 82.75% of all collected mosquitoes. One species in particular, Culex sitiens, was over-dominant (n = 1874) representing 60% of all collections. This species was present in all sites studied, but occurred only during the rainy season. The second most abundant species, Culex vishnui, representing 15% of all collected species (n = 472 specimens), was also present in all the sites, but was found throughout the year.

The core of the reserve, Koh Sralau, located in the mangrove forest, harbored the highest Culicidae diversity with 32 species (Table 1). The lowest diversity was observed in Koh Moul, the site located near the beach, with only 7 species (Table 1). The diversity and number of collected mosquitoes were the highest during the rainy season: during the dry season, only 14 species and 345 specimens were collected, whereas 29 species and 2762 specimens were observed during the rainy season (Table 1).

Only 8 species could be found through the year: Aedes albopictus, Anopheles baezai, Armigeres subalbatus, Coquillettidia crassipes, Culex bitaeniorhynchus, Cx. vishnui, Mansonia dives, and Mimomyia hybrida. Except the last 2, all of these species are considered to be of medical importance.

During our collection, a total of 12 species from 5 different genera are reported to be of medical importance (Table 2) and represented 77.9% (n = 2423) of all collected mosquitoes.

The present study provides—to our knowledge—the first list of mosquitoes in the mangrove forests of Koh Kong. Besides providing a baseline for future researches in this conserved area, it is another step forward in assessing the local risks of mosquito-borne disease.

Of the 290 known species recorded from Cambodia [26], the mosquitoes sampled in Koh Kong, with 37 recorded species, represent 12.7% of the Cambodian fauna. The Culicidae fauna of Koh Kong is quite diverse compared with that in the mangrove forest at the mouth of the Bangpakong river in Thailand, where only 14 species were recorded [33]. The most extensive work in the southern Asian region has been conducted in the Indian mangrove forests, where 43 species were recorded in Bhitarkanika (Orissa; [6]), 19 in the Sunderbans (West Bengal; [7]), 53 in the Andaman Islands [8], and 12 species in Coringa (Andhra Pradesh; [9]). Typically, mangrove forests, despite having a low plant diversity, are insect-species rich, having a very adapted fauna [5].

Most of the species collected belonged to the genus Culex, where 11 species were recorded. One species in particular, Cx. sitiens, is over-dominant during the rainy season, representing 60.3% of all collected mosquitoes (1874 specimens collected), and was collected across all sites. This result is not surprising because C. sitiens is strongly associated with mangrove forests [31], and is considered to be a saltmarsh mosquito [34, 35]. Easton [35] has made similar observations in mangroves in Macau, where this species was the second-most abundant, after Cx. quinquefasciatus. The latter species was only collected once in Koh Sralau during the rainy season. According to T Ismail et al. [15], Cx. sitiens was found to be predominant in the mangrove forest of Merbok (Malaysia), but in the present study, only after Verralina butleri, a species that was not observed in Koh Kong. The seasonality seems important for the members of the Culex genus, where only 6 species were collected during the rainy season.

In total, 5 species of Aedes were collected. All were collected (except Ae. albopictus) during the rainy season. Interestingly, despite a 5-year extensive collection through 15 provinces in Cambodia and the collection of more than 230,000 mosquitoes [26], Aedes (Lorrainea) amesii (n = 4) and Aedes (Rhinoskusea) longirostris (n = 30) were collected only in the Koh Kong mangrove forests. Both species are attracted to mangrove swamps [23, 36]. Immature stages of the subgenus Rhinoskusea are usually confined to brackish water coastal and river areas, and can be found in crab holes and small ground pools associated with mangroves [37]. The 2 Stegomyia species, Ae. albopictus and Ae. aegypti, have been reported from mangrove areas, being able to develop in brackish waters [38]. For the last Aedes species, Ae. (Phagomyia) prominens, very little is known about its biology [37], but this species was previously recorded in Cambodia in Mondulkiri province [26], suggesting that its distribution is not exclusive to mangrove ecosystems.

The genus Anopheles is poorly represented, with only 2 species recorded. According to Klein [39], An. baezai and An. indefinitus (named An. sundaicus in his article) are typical coastal Anopheles breeding in brackish waters on coastal regions. They are usually restricted to a narrow fringe along the coastline [39]. These data are consistent with our observations, where only 8 specimens were collected, mainly on Koh Sralau island and on the beach nearby.

The genera Mimomyia and Uranotaenia were present with 3 species for each genus. Both genera are typical saltmarsh mosquitoes. While very little is known about the biology of Mimomyia, the immature stages occur in swamps and marshes with dense vegetation. The adults of several species have been reported to bite humans, but none are considered to be serious pests [20]. Species of the genus Uranotaenia are known to bite mainly amphibians and reptiles, but birds and mammals can serve as secondary hosts. This genus is not known to be involved in any pathogen transmission [20]. The immature stages of Uranotaenia species utilize a wide range of habitats, including swamps and salt marshes [37].

Verrallina dux were collected only in Koh Sralau (the core of the reserve), and while the adults can be found resting in crab holes, its larvae can be found in temporary stagnant water holes, such as in footprints in rice fields, and are reported to be common in mangrove swamps in salt marshes and brackish waters [40]. Mansonia larvae occur generally in permanent waters and are associated with aquatic plants such as water lettuces (Pistia sp.). The larvae attach themselves to the floating roots of the plant to obtain oxygen from their aeriferous parenchyma. They can detach from and reattach to their host plants quite readily. Species of the genus Mansonia display the same biology and are also common in mangrove forests [35]. Females of several species in both genera are nocturnal biters, known to feed on humans and involved in the transmission of various arboviruses [37].

In total, 12 species of medical importance belonging to 5 different genera were collected, and represented 77.9% (n = 2423) of all collected mosquitoes. Most were collected during the rainy season.

Some 9 different species are vectors of Japanese encephalitis virus (JEV). The 2 main JEV vector species recognized are Culex tritaeniorhynchus and Cx. gelidus [30], both present in mangrove forests. The remaining 7 species are confirmed vectors: Ae. albopictus, Ar. subalbatus, Cx. fuscocephala, Cx. bitaeniorhynchus, Cx. sitiens, Cx. quinquefasciatus, and Cx. vishnui [30]. The JEV epidemiological cycle is undertaken through several hosts: wild birds, pigs, and mosquitoes, and is considered to be the most important cause of human encephalitis in Southeast Asia, and well established in Cambodia [12, 27, 30].

The 2 Stegomyia species (Ae. aegypti and Ae. albopictus) present in Koh Kong are considered to be potential vectors of dengue, Zika (along with Cx. quinquefasciatus), and chikungunya viruses [28]. Even though there is no evidence of intense circulation of West Nile virus (WNV) in Cambodia [41], concerns are arising about the ability for WNV to spread along bird migration routes based on its recent expansion in Eurasia [41]. This virus is naturally maintained in an enzootic transmission cycle of mosquitoes acting as vectors, and birds acting as the amplifying hosts. Humans and other mammals can serve as accidental/dead end hosts [42]. In Koh Kong, 2 more species are known vectors, namely Cx. quinquefasciatus and Mansonia uniformis [43]. All of them are known to be zoo–anthropogenic species [44, 45]. Blood meal analysis of Cx. quinquefasciatus demonstrated that they are highly ornithophilic besides feeding on mammals [46]; therefore, it is most likely that this vector can act as an important bridge vector and contribute to viral amplification [43].

Although only one specimen of parasite-carrying mosquitoes was captured, Anopheles indefinitus is reported to be potential vector of malaria [29, 47, 48, 49, 50] and 4 species are known to transmit filaria parasites, namely Ar. subalbatus and Cx. bitaeniorrhynchus transmitting Wucheria bancrofti, and Cx. sitiens and Cx. bitaeniorrhynchus transmitting Brugia malayi [49, 51]. However, these parasites were not yet recorded in Cambodia.

On a more general note, of the 12 medically important species, 3 of them were caught in each of the 3 sites. Cx. sitiens, a JEV vector, represents 60.3% of all collected mosquitoes and was found in all sites, but only during the rainy season. However, the 2 species Ae. albopictus and Cx. vishnui were found during both seasons. The first of these species is circulating at low intensity (representing 0.45% of all collected mosquitoes), but is a known vector of several arboviruses of high human medical importance [28]. The second species Cx. vishnui represents 15% of all collected mosquitoes in all habitats and almost all seasons. By contrast, in other sites in Cambodia, it usually accounts for more than half of the collected mosquitoes. Between 2017 and 2018, Boyer et al. [12] surveyed the mosquito population in 24 schools in Kampong Cham and Tboung Khmum provinces in Cambodia. Of 37,725 mosquitoes collected, 51.8% were Cx. vishnui. The over-dominance of this species was recorded in several other regions in Cambodia, such as in Prek Toal, where they represented 59.4% of all collected mosquitoes [52]. These 2 species, present throughout the sites, throughout the year, and across Cambodia, may represent a risk as bridge vectors for new or emergent zoonotic diseases. The rapid development of human settlements, and continued urbanization of ecosystems that are habitats for a rich diversity of wild fauna, represents a risk, as the dispersion of anthropophilic mosquito species into previously unfavorable habitats [53] could modify vector–host interactions. This could potentially lead to more contact with sylvatic reservoirs of zoonotic pathogens [54], and favors the emergence of new or neglected diseases in human populations.

Limitations of the present study include that each site was visited only once per season (rainy and dry) and that only BG and light-traps were used to collect mosquitoes. Increasing the diversity of traps and methods such as larvae collection are the best ways to improve the diversity of the collected Culicidae.