Mosquito (Diptera: Culicidae) Diversity and Community Structure in Doi Inthanon National Park, Northern Thailand

Simple Summary Mosquitoes are small flies, some of which are known as principal vectors of the pathogens that cause dengue, malaria, filariasis, chikungunya, Japanese encephalitis, and Zika in many countries of the world. However, the majority of the species are important components of ecosystems. We evaluated the diversity and community structure of mosquitoes in Doi Inthanon National Park, named for the highest mountain in Thailand. The park mainly consists of natural forest, but it hosts a number of human activities. A total of 140 mosquito species were identified among 3,795 specimens. The most dominant genera were Culex (34.3%), Aedes (19.3%), Anopheles (13.6%), Uranotaenia (10.7%), and Armigeres (5.8%). The five most abundant species were Ae. albopictus, Ae. vittatus, Cx. bitaeniorhynchus, Cx. mimulus, and Tripteroides aranoides. Species richness was higher in the rainy season than in the cold and hot seasons. Natural phytotelm habitats for immature stages had a higher diversity than artificial container habitats. A greater number of species were found in forest areas than in agricultural areas and villages. Ground pools, stream pools, rock pools, bamboo stumps, bamboo internodes, and rice fields were the most preferred natural habitats. Ground pools, rock pools, and stream margins, which had a high diversity index, were the most important habitats for mosquitoes in the park. Abstract Urbanization and human activities create new suitable aquatic habitats for the immature stages of mosquitoes in many countries. This also applies to Doi Inthanon National Park in northern Thailand, which is named for the highest mountain in the country. Despite its popularity, there is no information regarding mosquito diversity and community structure in the different ecosystems of the park. Monthly collections of immature stages from various habitats were conducted from August 2004 to December 2005 using dipping and sucking methods. The specimens collected from each habitat were reared to adults and identified based on their morphology. Diversity parameters and community structure were statistically analyzed. A total of 140 species (3795 specimens) belonging to 15 genera were identified. Among these, four genera (Culex, Aedes, Anopheles, and Uranotaenia) had high species richness, each represented by 48, 27, 19, and 15 species, respectively. Aedes albopictus was the most relatively abundant species, representing 6.7% of the total number of captured specimens, followed by Tripteroides aranoides (5.6%) and Cx. mimulus (5%). Species richness in natural habitats was significantly higher than in artificial containers. Species richness and abundance were highest in the rainy season. In comparison to agricultural areas and villages, mosquito diversity was found to be higher in forest areas. Ground pools, stream pools, rock pools, bamboo stumps, bamboo internode, and rice fields were the most preferred natural habitats. The results indicate that Doi Inthanon National Park has a high mosquito diversity. Each species exhibits differences in abundance and distribution in different habitats, which is useful information for planning conservation measures and vector control in the park.


Introduction
Mosquitoes are insects of major public health concern, mainly for the role of some species as vectors of pathogens of human and animal diseases, including the pathogens that cause dengue, chikungunya, Zika, Japanese encephalitis, West Nile fever, yellow fever, and malaria in humans. Approximately 60 pathogens can be transmitted to animals through mosquito bites [1][2][3]. Only female mosquitoes are of medical importance. Their bites can cause pain, itching, and red bumps, and scratching those bites can cause bacterial infections [4]. In 2020, there were 105,088 cases of the four main diseases in Thailand, i.e., dengue fever (90,915 cases), malaria (4473 cases), chikungunya fever (14,100 cases), and Zika fever (155 cases) [5]. However, not all mosquitoes are vectors (less than 5% of all mosquitoes in the world), and most of them are important components in aquatic and terrestrial ecosystems [4,6,7]. For example, nonbiting mosquitoes in the genus Toxorhynchites are beneficial because they can be used for biological control and to study virus propagation. The larval stages of Toxorhynchites are natural predators of other mosquito larvae (e.g., Aedes aegypti, Ae. albopictus, Culex quinquefasciatus, and Cx. vishnui) and aquatic organisms that live in the same phytotelm habitats [3,8]. In addition, Ae. aegypti, Ae. communis, and Ae. canadensis play a role as pollinators of Platanthera obtusata (an orchid) while feeding on the nectar of the flowers [9][10][11][12].
Doi Inthanon National Park is well known as "the roof of Thailand". The park is named for the highest mountain in the country (elevation 2565 m), and covers an area of 482.4 km 2 . It is a far eastern part of the Himalayan mountain range, with 2000 mm of annual precipitation and an average annual temperature of 20 • C (0-28 • C) [49]. The park has diverse microhabitats and unique climates depending on elevation, which ranges between 400 and 2,565 m above sea level. Most areas of higher elevation include several forest types, especially cloud forest, which has high moisture throughout the year. There are numerous plant species and the animal fauna includes 65 species of mammals and 370 bird species [49][50][51][52], and many groups of insects [53][54][55][56][57]. The pa'rk has numerous permanent and seasonally temporary streams and diverse other aquatic habitats, which serve as natural habitats for the immature stages of mosquitoes [4,58]. However, human activities, including urbanization, agriculture, and tourism, are the main cause of decreasing natural habitats, while new habitats are being created, some especially suitable for vector species [59][60][61][62][63][64][65].
Due to a lack of data on the diversity and community structure of mosquitoes in tropical forest ecosystems in Thailand, this study aimed to evaluate the diversity and community structure of mosquitoes across different ecosystems in Doi Inthanon National Park.

Mosquito Collections
The immature stages of mosquitoes (larvae and pupae) were collected monthly from various aquatic habitats from August 2004 to December 2005 using standard dipping and sucking methods. The larvae collected from each site were kept in Whirl-pack bags (width 7 cm, length 17 cm, BioQuip Products, Inc., Compton, CA, USA) containing 100 ml of water from the habitat. The collections were transported to the laboratory and the immature stages of each were reared in individual aluminum bowls. The third-and fourth-instar larvae were separated into individual plastic tubes (2.9 × 8.3 cm) and reared to obtain adult specimens. Emerged adults were pinned, and their associated larval and/or pupal exuviae, along with some fourth-instar larvae from the same collection, were mounted on microscopic slides. Some specimens were also preserved in 75% ethanol.

Larval Mosquito Habitats
During field surveys, each larval habitat was classified as belonging to one of three types of ecosystems, i.e., forest, village, and agriculture. The specific habitats were classified into 31 types in two categories, natural aquatic habitats (animal footprint, bamboo stump, banana stump, bog, cave pool, rock hole, tree hole, ground pool, rice field, etc.) and artificial (human-made) container habitats (bottle, cement tank, jar, tire, etc.) ( Table S2). Details of each habitat type were described in Rattanarithikul et al. [23].

Statistical Analyses
Species richness and abundance of the mosquitoes were recorded for each habitat type. Percentages of relative abundance of each habitat type were calculated by dividing the total number of species that were found in the habitat by the total number of specimens collected.
The percentage of species occurrence (%SO) was calculated by dividing the number of habitat types where a species was taken by the total number of habitat types (n = 31). Diversity index analyses for all specimens collected are Shannon_H, Simpson_1-D, Domi-nance_D, and Evenness_eˆH/S. Detrended correspondence analysis (DCA) was used to analyze community structure and distribution of the mosquito species associated with each habitat type. All data were analyzed using PAST version 4.07 [73]. To compare mosquito diversity with different sample sizes in each community and sampling sufficiency, species accumulation rarefaction and extrapolation curves were generated using the iNEXT software (Online Version 2022). Comparisons between the natural habitats and artificial containers (human-made), and between habitat types (forest, agriculture area, and village) and seasonal diversity were carried out with 1000 randomizations without replacement and at a 95% confident interval [74].
Genus Lutzia. Three species in the genus Lutzia were collected. Lutzia chiangmaiensis was the most abundant, representing 64.7% of the Lutzia identified, followed by Lt. Other genera. Data for seven other genera, for which relatively few specimens were collected, including Coquillettidia, Heizmannia, Mimomyia, Orthopodomyia, Toxorhynchites, Tripteroides, and Verrallina, are provided in Table S2.

Diversity Comparisons
Seasonal species richness and abundance of mosquitoes revealed distinct differences between seasons, with peaked abundance in the rainy season, followed by the cold and hot seasons (Figure 7).
Sample coverage of each season was high in all seasons (rainy = 0.993, cold = 0.991, hot = 0.994), indicating that the sampling in this study was efficient. Diversity data of mosquitoes collected during each season are shown in Table 1. A total of 32 species (23% of all species collected) were encountered throughout the year, whereas 74 species (53%) were found in only one season, in which their activity was dependent. Interestingly, the most relatively abundant species, Ae. (Stegomyia) albopictus, was found during the rainy (peak) and cold seasons but was absent in the hot season.  Species diversity of mosquitoes in natural and artificial containers is compared in Table S2. The accumulation species richness curve indicated that the number of mosquito species in natural habitats (138) was significantly higher than in artificial containers (33) (t = 23.30, df = 442.620, p < 0.001) (Figure 8). The diversity data for mosquitoes in both natural and artificial habitats are presented in Table 1. Mosquito diversity in forest, agriculture, and village areas was compared. The species richness accumulation curve revealed that the forest area (108) had significantly more mosquito species than the agriculture (88) and village (57) areas (F = 4.125, df = 151.2, p = 0.018) (Figure 9). The diversity data for mosquitoes in each community are shown in Table 1.

Aquatic Habitat Preferences
The most preferred natural habitats for mosquito species were ground pools, stream pools, and rock pools, which supported 55, 39, and 37 species, respectively. Cement tank, tire, bottle, and water-bucket sites were the most preferred artificial habitats, supporting 17, 11, 8, and 8 species, respectively ( Figure 10, Table S2). The diversity index (Shannon_H index) was highest for the ground pool habitats, followed by the rock pool, stream margin, stream pool, and tree-hole habitats, with diversity indices of 3.47, 3.09, 2.94, 2.93, and 2.74, respectively (Table S2).

Correlation of Mosquito Species with Habitat Types
Detrended correspondence analysis indicated that the distribution of species of each genus of mosquitoes correlated with habitat types (Figure 11). Mosquitoes of the genus Culex were found in 93.5% (29/31) of all habitat types. Most Culex species were associated with ground pools, stream pools, rock pools, stream margins, and bamboo stumps, which harbored 29, 19, 17, 16, and 11 species, respectively (Figure 11a). The most widely distributed and abundant species, Cx. (Culex) mimulus, was positive in 16 habitat types, with high abundance observed in ground pools, rock pools, and stream pools, but it was also found in a wallow, leaf axils, a swamp, and a jar. Culex (Culiciomyia) sasai was found in 35.5% (11/31) of all habitat types, with high abundance in cement tanks and fallen leaves, but it was also found in stream pools and bamboo internodes. Culex (Lophoceraomyia) sp. 4 was found in 35.5% of all habitat types, with high abundance in the rock pool and ground pool habitats. Additionally, some specimens of this species were found in tree holes, bamboo stumps, and plastic sheets. Culex (Oculeomyia) bitaeniorhynchus was found in 23.6% (7/31) of all habitat types, the occurrence of which was strongly correlated with stream pool and rice field habitats. The medically important species Cx. (Cux.) quinquefasciatus was found only in artificial container habitats (tires and cement tanks). The remaining 15 Culex species were restricted to a single habitat type. For example, Cx. (Cui.) harrisoni was found only in a cave pool, Cx. (Cux.) barraudi in ground pools, and Cx. (Ocu.) sinensis in rice fields (Table S2).
Mosquitoes of the genus Aedes were found in 83.9% (26/31) of all habitat types, and most of those species were found in bamboo internodes, tree holes, and stream pools (Figure 11b). The most encountered species (most occurrence records), Ae. (Hulecoeteomyia) harveyi, was found in 61.5% (16/26) of all habitat types, with high abundance in water bucket, banana stumps, bamboo internodes, bamboo stumps, and coconut shells. This species was also found in various other habitat types, such as tree holes, cement tanks, stream pools, animal footprints, and crab holes. The most abundant species, Ae. (Stg.) albopictus (a medically important species), was found in 53.8% (14/26) of all habitat types. High numbers of this species were collected from the bamboo stumps, bamboo internodes, tires, rock holes, and a water bucket. The remaining five species of Aedes collected were specific to various habitat types, such as cave pool for Ae. (Borichinda) cavernicola, tree hole for Ae. (Downsiomyia) albolateralis, and bamboo stump for Ae. (Dow.) novoniveus (only one specimen) ( Table S2).
Mosquitoes of the genus Anopheles were found in 51.6% (16/31) of all habitat types, and most species were found in ground pools, stream pools, and rice fields (Figure 11c). Anopheles (Ano.) cameronensis was found in the greatest number of habitat types, having been found in 50% (8/16) of all types. The highest density of this species was recorded in ground pools, stream margins, ditches, stream pools, and a bog located at high elevation (2520 m above sea level). Anopheles (Ano.) bengalensis was the most abundant species, which was found in 7/16 (43.8%) of all habitat types. It was strongly associated with stream pools, ground pools, and stream margins. Among malaria vectors in Thailand, An. (Cel.) minimus was found in stream pools, stream margins, and ground pools in high densities, and some specimens were found in crab holes, swamps, and rice fields. The great numbers of An. (Cel.) maculatus were recorded in ground pools and rock pools, and one specimen was found in a bamboo internode. Most specimens of An. (Cel.) sawadwongporni were found in rock pools and stream margins but they were also found in a cement tank. Anopheles (Cel.) dirus was only found in animal footprints. Only one specimen each of An. (Cel.) nivipes, An. (Cel.) pseudowillmori, and An. (Cel.) vagus was found in a ground pool (Table S2).  Table S2.
Members of the genus Uranotaenia were found in 48.4% (15/31) of all habitat types, and most species were collected from ground pools, rock pools, and stream pools (Figure 11d, Table S2). Uranotaenia (Ura.) sombooni was the most widely distributed species, found in ground pools and flood pools. Some specimens were found in a stream pool and stream margin, and one specimen was found in a bamboo internode. Uranotaenia (Ura.) annandalei was found in a stream margin and stream pool.
Six species of the genus Topomyia were collected from leaf axils, while some were found in fallen leaves, tree holes, and bamboo internodes. Species of the genus Toxorhynchites were mostly found in bamboo stumps, bamboo internodes, tree holes, and bottles. The most abundant species, Tx. (Toxorhynchites) gravelyi, was found in bamboo stumps, while Tx. (Tox.) splendens was found only in tree holes and Tx. (Tox.) leicesteria only in bamboo internodes. Three species of the genus Tripteroides were found in bamboo internodes, bamboo stumps, and tree holes but most were found in bamboo internodes.
The species of six genera for which the fewest specimens were collected were each restricted in only one habitat type. For instance, Cq. (Coquillettidia) crassipes were found only in ponds, Hz. (Heizmannia) reidi and Or. anopheloides in tree holes, Ml. genurostris and Ml. jacobsoni in leaf axils, one specimen of Mi. (Eto.) luzonensis in an animal footprint, and Ver. sp. 1 in ground pools (Table S2).

Discussion
The present study indicates that Doi Inthanon National Park is an important area for mosquito diversity. The park has high species richness and abundance of mosquitoes representing one third of the total number of species recorded in Thailand [4]. The topographic complexity and variety of aquatic sources in the park are available for mosquitoes throughout the year [49,57,75]. The great diversity of mosquitoes (140 species in 15 genera) collected in this study is consistent with previous reports. For example, 160 species in 18 genera were found in the Amazon rainforest [76], 117 species in 17 genera in the northwestern Amazon [35], and 103 species in 16 genera in Cantareira State Park in Brazil [77]. However, low numbers of species can be affected by different collection methods, as seen in some previous studies, such as 26 species in five genera collected in Vietnam [78], 50 species in 12 genera in the Amazon basin, Peru [79], 82 species in the Caatinga biome, Brazil [80], 20 species (10,131 specimens) from Amazonas State, Brazil [37], and 103 species in 16 genera in Cantareira State Park, Brazil [77]. According to these data, it is obvious that the tropical region is an area with high levels of mosquito diversity. However, the diversity, distribution, and abundance of mosquitoes are not only dependent on suitable habitats, but also on physiochemical factors in those habitats that influence larval occurrence [7,[81][82][83].
Findings for the dominant genus Culex in this study correspond with accounts in previous reports [77,79,80]. Most species in this genus have high abundance and richness in forest areas [67,84]. Our results showed that Culex larvae were found in various types of habitats, which is similar to the previous findings reported by [7,27,67,85]. Doi Inthanon National Park has environmental heterogeneity that supports a variety of habitats for Culex mosquitoes, especially ground pools, rock pools, and rice fields in the rainy season. Similarly, some studies have found that the majority of habitat types utilized by Culex are semi-permanent or permanent bodies of fresh, brackish, clean, or heavily polluted water, and several species develop in leaf axils, tree holes, rock holes, crab holes, and other small collections of water [86,87]. As in another study, Cx. (Cux.) mimulus, the most abundant Culex species in the present study, was mostly found in ground pools and rock pools, but specimens were occasionally found in other habitats, including an animal wallow, leaf axils, tree holes, and a jar [67]. In addition, our data for Cx. (Cux.) quinquefasciatus are consistent with the previous report by Almirón and Brewer [88], who also found this species more frequently in artificial containers (tires, tank in disuse, and cistern). The habitats of 27 species of Aedes collected in the present study are accordant with those recorded by Rattanarithikul et al. [14,23], which included tree holes, bamboo stumps, and bamboo internodes as the principal habitats for Aedes larvae in Thailand. Aedes (Stg.) albopictus occupied the widest variety of habitats, especially phytotelmata and artificial containers, with a higher abundance in natural containers than in artificial containers. This finding agrees with the findings of studies conducted in several countries (Thailand, Brazil, Malaysia, Vietnam, China, and USA) in which Ae. albopictus also occurs in both natural and artificial containers [14,[89][90][91][92][93][94]. However, those studies were conducted in the rural and suburban areas, and a higher abundance in artificial containers than in natural containers was observed. For example, Vanwambeke et al. [89] found Ae. albopictus mainly in rural and peri-urban areas, where it was found in natural and artificial containers, with the latter making up 87% of collections.
We found 19 species of Anopheles in Doi Inthanon National Park, approximately one fourth of the 83 species recorded in Thailand [18,19,22,95]. Comparison of our findings with those of other studies confirms the high diversity of Anopheles in Doi Inthanon National Park. For example, 23 species were recorded in Indonesia based on collections using animal bait and animal-baited trap nets in three ecosystems, i.e., forest, non-forest, and coastal areas [96], 20 species collected by CDC light trap without bait at the China-Myanmar border [34], and 9 and 10 species collected by standard dipping in the Himalayan region and Ethiopia, respectively [7,97]. The larval habitats for species of this genus were strongly associated with a variety of forest types and clean water [22]. Most Anopheles larvae were found in ground pools, stream pools, and rice fields, which is in agreement with previous studies that revealed the typical habitats of the immature stages of anopheline species, which are usually found in clean, fresh, still, or slow-moving waters, and groundwater with abundant floating vegetation. However, some species are restricted to plant cavities in egg-cup-size pools in primary forests in northern Vietnam [98,99]. In a previous study [100], An. (Ano.) bengalensis, the most abundant Anopheles species in the present study, was found in a shaded pool beside a stream in a hilly and mountainous area similar to our finding of this species in Doi Inthanon National Park, where the species was mostly found in ground pools, stream pools, and stream margins. Additionally, this common species has been found in several types of habitats in natural forests, including cave holes, rock pools, crab holes, and animal footprints [22]. Anopheles (Ano.) cameronensis was mostly found in ground pools, stream margins, and bog in forest areas at elevations ranging between 1200 m and the summit of Doi Inthanon (2500 m), as reported by other researchers [22,100]. However, the dominance and abundance of mosquito species varied depending on collection methods and in different ecosystems [7,40,46,48,96]. Interestingly, one specimen each of An. (Ano.) sinensis and An. (Cel.) maculatus was found in a bamboo internode despite commonly occurring in rice fields, grassy ponds, ditches, springs, seepages, and small streams partly exposed to sunlight [99]. Therefore, changes in environment and ecology, and the effect of human activities are affecting the mosquito community, leading to adaptation to new aquatic habitats. Thus, regular entomological evaluation is essential for vector monitoring.
Based on the results of the present study, the seasonal diversity and abundance of mosquitoes in Doi Inthanon National Park varies depending on the species, some of which are present throughout the year. However, in general, species richness and abundance peak in the rainy season and decrease in the cold and hot seasons, in agreement with the finding of other studies conducted in the Himalayan region [7], Ghana [101], and Mexico [102]. Several studies conducted on vector species in Thailand have shown that species richness and populations exhibit high fluctuation in the rainy season [39,42,43,47]. Rainfall is the key factor that influences the diversity and distribution of mosquitoes.
While comparing the diversity of mosquito species in natural and artificial habitats, it was discovered, as expected, that natural habitats support many more species than artificial habitats. Based on a comparison of the findings of this study with those of previous studies, it is obvious that there is a high diversity of species in forests, as opposed to low diversity but higher abundance in areas outside of forests, particularly in artificial habitats [37,96,103]. Doi Inthanon National Park is primarily comprised of forested areas that provide a variety of habitats for various mosquito species, whereas artificial habitats are generally found in villages, agricultural areas, and some tourist sites, which are minor areas of the park and are unsuitable for many species other than Ae. (Stg.) albopictus, which is abundant in such areas.

Conclusions
Based on the results of this study, it is apparent that Doi Inthanon National Park has very diverse populations of mosquitoes. Culex species and Ae. (Stg.) albopictus are present in highest abundance. Species richness is highest in the rainy season, much lower in the cold and hot seasons, and natural habitats harbor more species than artificial habitats. Forests host a greater diversity of species than agricultural and village areas. Ground pools, stream pools, and rock pools are important habitats for the immature stages of mosquitoes in the park. Information on the distributions and seasonal activity of mosquitoes obtained in this study is important for planning programs for the control of vector species.