Biodiversity, seasonal abundance, and distribution of blackflies (Diptera: Simuliidae) in six different regions of Thailand

Blackflies are an important medical and veterinary group of small blood-sucking insects. Ninety-three blackfly species have been reported in Thailand. However, information on their biodiversity and population dynamics in each region is lacking. The main aim of this study was to assess the regional biodiversity, seasonal abundance and distribution of blackflies in six eco-geographically different regions in the country. Blackfly larvae and pupae were sampled monthly from 58 sites between May 2011 and April 2013. Diversity parameters, seasonal abundance, regional distribution and frequency of species occurrence in stream sites were analyzed. A total of 19,456 mature larvae representing 57 species, and belonging to six subgenera in the genus Simulium Latreille (s.l.), were found. The five predominant taxa were S. fenestratum (8.6%), the S. asakoae complex (8.3%), S. nakhonense (7.5%), the S. siamense complex (7.4%) and the S. doipuiense complex (6.7%). The most frequent taxa at all sites were the S. asakoae complex (84.5%), followed by S. fenestratum (82.8%), the S. siamense complex (75.9%), S. decuplum (60.3%), S. nakhonense (58.6%) and the S. tani complex (48.3%). The richness of regional species was highest (40 species) in the north and predominated in the cold season. However, blackflies in the south predominated during the hot season. The highest numbers of blackflies collected from central, northeastern, eastern and western regions of the country were observed in the rainy season. Overall, the mean number of blackflies collected across the six regions during the rainy and cold season had no statistically significant difference, but it differed significantly in the hot season. Blackflies in Thailand were surveyed in all three seasons across six geographical regions. These findings demonstrated that blackfly communities at each stream site varied with seasonality, and the regional relative abundance of blackflies differed markedly in the hot season. It was also found that the occurrence and distribution of blackflies in each region were associated strongly with elevation.


Background
Blackflies (Diptera: Simuliidae) are distributed widely in all zoogeographical regions and found almost everywhere with running water that is suitable as a habitat for their aquatic stages [1]. Larvae and pupae are aquatic, and attach themselves to various submerged objects in many types of lotic environments, ranging from large rivers to tiny spring-fed trickles, and from swift currents to water that barely moves [2]. The choice of habitat usually varies between species. Due to their bloodsucking habits, adult females of certain blackfly species are of a medical and veterinary importance. Blackflies have been considered as vectors of many pathogens, such as filarioid nematodes of the genus Onchocerca in humans, cattle and deer, the genus Dirofilaria in bears, the genus Splendidofilaria in ducks; blood protozoans of the genera Leucocytozoon and Trypanosoma in birds; and viruses (rift valley fever, vesicular stomatitis) in horses and cattle; as well as chlamydial bacteria that cause blindness in sheep and abortion in cattle [1][2][3]. Furthermore, blackfly bites can cause other severe problems in humans, since they frequently inflict pain, localized swelling, chronic dermatitis and inflammation accompanied by intense irritation that lasts for several days or even weeks [2].
In Thailand, a total of 93 blackfly species belonging to six subgenera, including Asiosimulium, Daviesellum, Gomphostilbia, Montisimulium, Nevermannia and Simulium, have been reported ( [4], W. Srisuka, unpublished observations), with most new blackfly species being discovered in the northern part of the country. Remarkably, although the above information reflects rich species diversity, there are only a few reports of simuliids from other regions in Thailand, for instance, S. otsukai, S. thongsahuani, S. datfaense and S. trangense, in the south [5][6][7]; S. vanellum from the west [8], S. atipornae and S. lomkaoense from central Thailand [9,10]; and S. kuvangkadilokae from the northeast [11,12]. Notably, there were no reports on regional biodiversity, seasonal abundance or distribution of blackflies in macro-scale areas of Thailand, apart from only the hotspot area in the tropical rainforest at Doi Pha Hom Pok National Park, in the northern region [13]. Additionally, human-biting blackfly species are found in large numbers, and cause irritation in domestic environments and to indigenous people and tourists [14], thus, most previous studies focused on the annual biting activity of adult females at Doi Inthanon and Doi Suthep-Pui National Park as well as in the village of Ban Pang Faen, Chiang Mai Province, northern Thailand [14][15][16][17].
Hence, the main aim of this study was to determine the seasonal abundance and dynamics of blackflies in six geographically and ecologically different regions of Thailand.

Study areas and sampling
This study was carried out at 58 fixed-stream sites in 41 provinces in six regions across Thailand, including 15, 10, 10, 7, 8 and 8 sites in the north (9 provinces), central (7 provinces), northeast (7 provinces), east (5 provinces), west (5 provinces) and south (8 provinces), respectively ( Fig. 1, Additional file 1: Table S1). A total of 696 collections were made in this study (12 at each 58 fixedstream sites at monthly intervals) from May 2011 to April 2013 which covered all seasons for each region. Larvae and pupae were hand sampled using fine forceps from available substrates in streams, such as fallen leaves, mud or rock surfaces, and trailing grasses. Fortyfive minutes exactly were spent for the collection of larvae and pupae by the same person (one person) at each stream site. Larvae were preserved in 80% ethanol. The substrates were cut into pieces so that each part harboured a single pupa. Matured pupae were maintained individually in a plastic tube (10 cm long and 1.7 cm in diameter) with very little water at the bottom until adults emerged. After emergence, adult flies were kept alive in the same tube for at least 24 h, to secure hardening and colouring of their body and legs. Adult flies, associated with their pupal exuviae, were used to confirm the species identification of the larvae.

Meteorology and regions
The classification of the season in each region follows the Thai Meteorological Department, which bases its records on rainfall and air temperature data. Thus, each year is divided climatically into three seasons in the following regions: North region, with hilly and mountainous areas ranging from 392 to 2210 m in height, total rainfall of approximate 1287 mm and an average air temperature of 25°C; West region, with mostly mountainous areas in the range of 111-560 m in height, similar to the north, and total rainfall and average air temperature of 1243 mm and 27.13°C, respectively; Central region, with largely low-level plains and a few mountains ranging from 167 to 1550 m high in the northern and western part of the region, with a total rainfall and average air temperature of 1377 mm and 26.1°C, respectively; Northeast region, which is a naturally high-level plain called the northeast plateau; Northwest-southeast region, which has Phu Phan Ridge oriented in the northeastern portion that separates this area into two basins, the first one is a large high-level plain in the west and the other smaller and sloped towards the east with an elevation ranging from 110 to 1337 m; this region has a total rainfall and average air temperature of 1589 mm and 25.1°C, respectively; East region, which is mountainous in the northeast, and its eastern area is close to the Gulf of Thailand with an elevation ranging from 76 to 409 m, and total rainfall and an average air temperature of 2903 mm and 26.7°C, respectively; South region, which has a peninsula mountain spine that is very steep. Its eastern area is close to the Gulf of Thailand and its western region near the Andaman Sea. It has an elevation ranging from 111 to 560 m, and total rainfall and an average air temperature of 2601 mm and 28.9°C, respectively.

Data analysis
Species and relative abundance of mature larvae at each site were recorded. The frequency of blackfly species was calculated by the total number of species occurrence divided by the total number of collections and presented in percentages. Stream occurrence (SO) (expressed in percentage) was obtained by calculating the number of sites where a species was taken and dividing it by the total number of sites sampled (n = 58). Species diversity and richness (SDR) version 4 [27] and PAST version 3.11 were employed for statistical analyses [28]. Determination and comparison of diversity parameters between regions were calculated using the Shannon-Wiener index (H), the expected value of H (Exp H) and evenness (J'). The species accumulation curves (rarefaction) for regions were also compared. In evaluating species richness, the first order jackknife was used to estimate the number of species presenting in all stream sites. Sample interpopulation was used to estimate the number of species from all regions (696 collections) [27]. Detrended correspondence analysis (DCA) was used to describe the regional distribution of blackfly larvae associated with sampling sites [28]. A ternary plot, based on data of blackfly species in each season and region, was used to interpret seasonal occurrence and abundance [28]. Regional and seasonal differences in blackflies were compared using non-parametric Kruskal-Wallis tests, and P-values were adjusted by the Bonferroni correction for post-hoc multiple comparison tests. In addition, the Mann-Whitney U-test was used to compare the mean number of blackflies collected from the southern region in two seasons. Statistical analyses were conducted using IBM SPSS statistics, version 24 for Windows (Chicago, SPSS Inc.). Statistical significance was set at P < 0.05.

Species diversity, richness and distribution pattern
Species diversity and richness of blackflies in each region are shown in Fig. 2, with the highest in the northern region (H = 3.1, J' = 0.8) and lowest in the southern (H = 2.1, J' = 0.5). Of 58 stream sites, the Shannon diversity index (H) was highest at Rom Klao (Phitsanulok Province), followed by Doi Phu Kha (Nan Province), Phu Ruea (Loei Province) and Mae Wong (Kamphaeng Phet Province), which represented 2.4, 2.4, 2.3 and 2.3, respectively (Additional file 2: Table S2).
The species accumulation (rarefaction) curves (Fig. 3) showed the expected and observed richness of species occurring in all sites, with a total from all collections being 71 and 57 species, respectively. The expected (± SE) and observed species richness were 52 ± 4.5 and 40 spp. in northern, 45 ± 4.1 and 34 spp. in the central, 38 ± 2.9 and 28 spp. in western, 31 ± 2.8 and 22 spp. in northeastern, 25 ± 4.3 and 17 spp. in southern and 24 ± 1.8 and 15 spp. in eastern regions, respectively (Fig. 4).

Seasonal dynamics
Overall, S. fenestratum was the dominant species during all three seasons (Additional file 3: Table S3). Almost all of the species in the north were dominant in the cold season, and S. chumpornense and S. phayaoense were recorded only during that time, while S. pahangense was collected only during the rainy season (Fig. 6, Additional file 3: Table S3). Most species in the central region were discovered in the rainy season, and S. oblongum, S. aureohirtum, S. bullatum, S. phukaense and the S. tani complex were found only during that time, whereas S. yongi was only recorded in the cold season (Fig. 6, Additional file 3: Table S3). A greater number of blackflies were collected during the rainy season in the northeastern, eastern and western regions (Figs. 7, 8, Additional file 3: Table S3). Simulium oblongum, S. aureohirtum and S. yuphae were found only during the rainy season in the eastern region, similar to species found in the central region. Remarkably, blackflies in the southern region were more dominant during the hot season (Fig. 8, Additional file 3: Table S3). The mean number of blackflies collected across the six regions during the rainy (Kruskal-Wallis test, H = 6.242, df = 5, P = 0.283) and cold season (Kruskal-Wallis test, H = 8.650, df = 4, P = 0.070) had no statistically significant difference, but it differed significantly in the hot season (Kruskal-Wallis test, H = 26.589, df = 5, P < 0.0001).

Eastern region
The most frequent taxa at all sites were S. decuplum, S. dentistylum, the S. siamense complex, S. fenestratum and the S. tani complex (100%), followed by the S.  Table S7). The mean number of blackflies captured in the hot season was lower than that in the rainy (Kruskal-Wallis test, H = 21.195, df = 2, P = 0.021) and cold season (Kruskal-Wallis test, H = 34.122, df = 2, P < 0.0001). There was a significant difference in a mean number of the blackflies collected in this region between the hot and rainy seasons (Kruskal-Wallis test, H = 15.033, df = 2, P < 0.005).

Discussion
Species composition, species richness, seasonal abundance and diversity The number of blackflies reached its highest during the cold season, according to a previous report by Srisuka et al. [13], who studied the seasonal biodiversity of blackflies at Doi Pha Hom Pok, northern Thailand. The greatest number of blackflies in the southern region was in the hot season. This study found that seven of seventeen species identified from this region increased their populations approximately two to three times during this season. This observation agrees with the study of blackflies in northern Sweden, where they were higher in the summer than other seasons [29]. The highest number of blackflies collected from central, northeastern, eastern and western regions of Thailand peaked in the rainy season. The findings in this study were consistent with those in a previous report by Pramual & Wongpakam [30], who studied the seasonal variation of blackflies at Phu Phan mountain range in northeastern Thailand. They demonstrated that the species abundance was higher in rainy seasons than in others and blackfly communities at each stream site varied with seasonality, i.e. S. nakhonense, the S. angulistylum complex and S. kuvangkadilokae were more dominant in the rainy season, whereas the S. asakoae complex, S. aureohirtum and S. trangense were dominant in the hot and cold season [30]. Likewise, blackflies were caught in higher numbers during the rainy season in Nigeria, Africa [31]. Takaoka [32] showed that seasonal abundance patterns of adult populations of S. ochraceum, the vector of onchocerciasis in Guatemala, Central America, differ by localities depending on the availability of permanent and temporary streams suitable for its immature stages. In addition to seasons and geographical locations, elevation also can influence blackfly populations. The results of this study showed that the Shannon diversity index was highest in areas with high elevations, i.e. Rom Klao (1047 m), Doi Phu Kha (1629 m), Phu Ruea (1337 m) and Mae Wong (1274 m). It was found that 36 species manifested in optimal or unique environments that had suitable factors for their breeding habitats. For example, S. baimaii breeds at Phu Kradueng, Loei Province in only high mountains, with slow-flowing streams exposed to sunlight. Likewise, all species members in the subgenus Montisimulium are restricted to high elevations at Doi Inthanon National Park, Chiang Mai Province. In contrast, S. gombakense has a wide vertical distribution range from a height of 500 m in small streams in the foothills to 2100 m near the summit of Doi Pha Hom Pok National Park [13], and it is also found at an elevation of 412 m at Mae Klang Waterfall, Doi Inthanon National Park [33]. In addition, the S. asakoae complex, S. fenestratum, the S. siamense complex, S. decuplum, S. nakhonense, and the S. tani complex were the most common taxa found in this study, which is similar to previous reports by Pramual & Kuvangkadilok [34], and Pramual & Wongpakam [30].

Relationship of subgenera to elevation
The subgenus Asiosimulium is a small and endemic subgenus in the Oriental region. It is represented by four species, of which three, S. oblongum, S. wanchaii and S. furvum, have been reported in Thailand [35][36][37], and the remaining one, S. suchitrae, in Nepal [38]. The first three species were found in lowland streams, flowing slowly over rock surfaces exposed to the sun during the rainy season, while S. suchitrae was found at high elevation (1826 m) in a small stream flowing slowly over rocks [38]. Both S. furvum and S. wanchaii were restricted to their sites, but S. oblongum was distributed widely in and near the northeastern, central and eastern regions. The subgenus Daviesellum is represented by two species, S. pahangense and S. courtneyi, in Thailand [39]. Only S. pahangense was distributed at high elevation from northern to central regions along the boundary with Myanmar, and also in lowland streams in the southern region. Most species of Gomphostilbia, such as those of the S. batoense, S. ceylonicum, S. epistum, S. gombakense and S. varicorne species-groups, are the second largest subgenus in Thailand and distributed at low elevations. This study found S. sheilae, the S. siamense complex and S. chumpornense in all six regions of Thailand, with their breeding habitats mostly in lowland streams as previously reported [7,30]. In contrast, species of the S. asakoae and S. darjeelingense speciesgroups were found in highland streams, except for the S. asakoae complex, which was distributed widely from low elevations to 2500 m at the summit of Doi Inthanon National Park, Chiang Mai Province and in other Asian countries, such as Malaysia, China (Hong Kong) and Vietnam [7,13,21,25,[40][41][42].
The subgenus Montisimulium is represented by six species in Thailand. Two of them, i.e. S. nanense and S. (Montisimulium) sp., were discovered in this study at high elevations, as reported by Takaoka & Somboon [43] and Takaoka et al. [44], who collected three species of this subgenus at high elevations ranging from 2229 to 3720 m in Bhutan and 1750 m in Vietnam, respectively. The remaining species have been found only on Doi Inthanon and Doi Pha Hom Pok in Chiang Mai Province [45,46]. Of ten species of the subgenus, Nevermannia reported in Thailand, seven were found in this study. Most species were collected at high elevations, for example, the high mountains of Chiang Mai Province, northern Thailand [13,26,47,48]. Other reports from several other Asian countries, such as Malaysia, Myanmar, Vietnam, Indonesia and Bhutan, indicated that members of this subgenus were associated with high elevations ranging from 1000 to 2532 m [43,[49][50][51][52][53]. The subgenus Simulium is the largest subgenus in Thailand, including 45 described species, of which 28 (62% of total species) were collected. Most of the common taxa, such as S. fenestratum, S. nakhonense, S. quinquestriatum, and the S. tani complex, breed in lowland streams. The findings of this study were in accordance with those reported by Takaoka et al. [44], Srisuka et al. [13] and Pramual & Wongpakham [30]. Species of the S. christophersi, S. malyschevi and S. variegatum speciesgroups were distributed in middle to high elevations (1200-2200 m), as previously studied in Vietnam and Thailand [44,54]. In contrast, most of the species within the S. multistriatum, S. nobile and S. striatum speciesgroups occurred in lowland streams, as reported by Srisuka et al. [13] and Ishii et al. [17]. Members of the S. griseifrons species-group colonized streams at low to high elevations (200-2210 m).

Conclusion
The findings of this study demonstrated that the richness and relative abundance of blackflies were different between regions, and blackfly communities at each