Biodiversity and Spatiotemporal Variations of Mecoptera in Thailand: Influences of Elevation and Climatic Factors.

Ecological analyses of the small and lesser-known insect order Mecoptera in Thailand are presented. Specimens were collected monthly over a period of 12 consecutive months, using both Malaise and pan traps, from 29 sampling sites located in 18 national parks throughout Thailand. A total of 21 species in four genera were identified from 797 specimens, including Panorpa (1 species), Neopanorpa (18 species), Bittacus (1 species), and Terrobittacus (1 species), with the latter genus representing a new genus record to Thailand. Neopanorpa harmandi, N. siamensis, N. byersi, and N. malaisei were the most abundant species, representing 27.4%, 11.3%, 10.3% and 8.8% of the total specimens, respectively. The species with the highest frequency, as indicated by the high percentage of species occurrence (%SO), was N. siamensis (51%), followed by N. byersi (34%), N. harmandi (34%), N. spatulata (27%), and N. inchoata (27%). Eleven species (52%) exhibited specific regional occurrences. N. tuberosa and N. siamensis had the widest distribution, being found in almost all regions except for western and southern regions for the first and second species, respectively. The seasonal species richness of Mecoptera was high during the rainy season in the northern, northeastern, central, eastern, and western regions, with the highest richness observed in July (15 species), followed by the hot (10 species) and cold seasons (7 species), while there was no significant difference in species richness between seasons in the southern region. Multiple regression models revealed a negative association between species richness and abundance of Mecoptera with both elevation and temperature, and a positive association between rainfall and species evenness. It is predicted that climatic changes will have a detrimental effect on the mecopteran community. The results of this study enhance the understanding of the ecological aspects of Mecoptera, offering crucial insights into its biodiversity and distribution, which are vital for conservation and forest management.

A new species of Simulium (Asiosimulium) (Diptera: Simuliidae) from northeastern Thailand, with its phylogenetic relationships with related species in the subgenus Asiosimulium.

Simulium (Asiosimulium) khongchiamense sp. nov. is described based on females, males, pupae, and mature larvae collected from Khong Chiam District, Ubon Ratchathani Province, northeastern Thailand. It is characterized in the female by the medium-long sensory vesicle, scutum with 3 dark longitudinal vittae and elongate cercus; in the male by the number of upper-eye (large) facets in 17 or 18 vertical rows and 18 or 19 horizontal rows, hind basitarsus moderately enlarged and ventral plate with the posterior margin moderately concave medially; in the pupa by the head and thoracic integument sparsely covered with tubercles and gill of arborescent type with 32 or 33 filaments; and in the larva by the postgenal cleft deep, reaching the posterior margin of the hypostoma and sheath of the subesophageal ganglion dark pigmented. DNA analysis based on COI gene of all known species of the subgenus Asiosimulium, except for S. shanense and S. suchitrae, indicated that this new species can be clearly differentiated from all other related species (S. phurueaense, S. oblongum, S. saeungae, S. furvum, and S. wanchaii) with interspecific genetic distances ranging between 4.79% and 19.18%. This is the eighth species of the subgenus Asiosimulium. Taxonomic notes are given to distinguish this new species from the 7 known species members in its same subgenus. Additionally, keys to species of all members in the subgenus Asiosimulium are provided.

Antennal morphology and sensilla ultrastructure of the malaria vectors, Anopheles maculatus and An. sawadwongporni (Diptera: Culicidae).

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (µm, mean ± SE) in the long sharp-tipped trichodea (40.62 ± 0.35 > 38.20 ± 0.36), blunt-tipped trichodea (20.39 ± 0.62 > 18.62 ± 0.35), and basiconica (7.84 ± 0.15 > 7.41 ± 0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97 ± 0.48; right: 33.27 ± 0.65) are also greater when compared to An. sawadwongporni (left: 30.40 ± 0.62; right: 29.97 ± 0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell.

Bacterial community structure of Anopheles hyrcanus group, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus from a malaria-endemic area in Thailand.

Bacterial content of mosquitoes has given rise to the development of innovative tools that influence and seek to control malaria transmission. This study identified the bacterial microbiota in field-collected female adults of the Anopheles hyrcanus group and three Anopheles species, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus, from an endemic area in the southeastern part of Ubon Ratchathani Province, northeastern Thailand, near the Lao PDR-Cambodia-Thailand border. A total of 17 DNA libraries were generated from pooled female Anopheles abdomen samples (10 abdomens/ sample). The mosquito microbiota was characterized through the analysis of DNA sequences from the V3-V4 regions of the 16S rRNA gene, and data were analyzed in QIIME2. A total of 3,442 bacterial ASVs were obtained, revealing differences in the microbiota both within the same species/group and between different species/group. Statistical difference in alpha diversity was observed between An. hyrcanus group and An. vagus and between An. nivipes and An. vagus, and beta diversity analyses showed that the bacterial community of An. vagus was the most dissimilar from other species. The most abundant bacteria belonged to the Proteobacteria phylum (48%-75%) in which Pseudomonas, Serratia, and Pantoea were predominant genera among four Anopheles species/group. However, the most significantly abundant genus observed in each Anopheles species/group was as follows: Staphylococcus in the An. hyrcanus group, Pantoea in the An. nivipes, Rosenbergiella in An. philippinensis, and Pseudomonas in An. vagus. Particularly, Pseudomonas sp. was highly abundant in all Anopheles species except An. nivipes. The present study provides the first study on the microbiota of four potential malaria vectors as a starting step towards understanding the role of the microbiota on mosquito biology and ultimately the development of potential tools for malaria control.

Simulium (Gomphostilbia) wijiti, a new species of black fly (Diptera: Simuliidae) from northern Thailand and its genetic position with related species in the Simulium ceylonicum species-group.

A new black fly species, Simulium (Gomphostilbia) wijiti, is described based on adult females, males, pupal exuviae and mature larvae from Mae Hong Son Province, Thailand. This new species is placed in the Simulium ceylonicum species-group. It is distinguished from four Thai members of the S. ceylonicum species-group [S. (G.) curtatum Jitklang et al., S. (G.) pangsidaense Takaoka, Srisuka & Saeung, S. (G.) sheilae Takaoka & Davies, and S. (G.) trangense Jitklang et al.], in the female by the short to medium long sensory vesicle; in the male by the large number of upper-eye (large) facets in 15 vertical columns and 15 or 16 horizontal rows; in the pupa by the dorsum of abdominal segments darkened; and in the larva by the antenna as long as or slightly shorter than the stem of the labral fan (longer than the stem of the labral fan in four other species). Phylogenetic analysis based on the COI gene sequences revealed that this new species is genetically closely related to S. leparense of the S. ceylonicum species-group, but is clearly separated from the latter species, and also from the three Thai related species (S. curtatum, S. sheilae and S. trangense) of the same species-group with interspecific genetic distances ranging from 9.65% to 12.67%. This is the fifth member of the S. ceylonicum species-group recorded from Thailand.

Discovery of a new black fly species in the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from northeastern Thailand and its phylogenetic relationships with seven Thai related species.

The female, male, pupa and mature larva of Simulium (Simulium) poolpholi sp. nov. collected from a large and seasonal stream in northeastern Thailand are described. This new species is placed in the S. striatum species-group by the female sternite 8 concave posteromedially in form of the inverted-U shape, ovipositor valve with a ventrally produced process along its inner margin, male ventral plate saddle-shaped, style with a basal protuberance, and pupal gill with 10 filaments. It is morphologically similar to S. (S.) phraense Takaoka, Srisuka & Saeung from northern Thailand by lacking annular ridges on the surface of pupal gill filaments but it is distinguished from the latter species by the arrangement of pupal gill filaments. Molecular analysis using the fast-evolving nuclear big zinc finger (BZF) gene shows that S. (S.) poolpholi sp. nov. formed a distinct clade, being separated from the seven other Thai species of this species-group by a genetic distance of 1.84-4.55%. Our discovery increases the number of species in the S. striatum species-group in Thailand to nine.

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

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.

Ultrastructure of sensilla on the antennae and maxillary palpi of the human-biting black flies, Simulium nigrogilvum and Simulium umphangense, (Diptera: Simuliidae) in Thailand.

Antennae and maxillary palpi are the most important sensory organs involved in the behaviors of black flies. The ultrastructure of sensilla on these sensory appendages of two human-biting black fly species, Similium nigrogilvum and Simulium umphangense, was studied for the first time. Wild adult females of both species were collected in Umphang District, Tak Province, western Thailand. The morphology and distribution of sensilla were examined using scanning electron microscopy. Overall, the morphology of the antennae and maxillary palpi and distribution of sensilla are similar in the two species. Four major types of sensilla were found on the antennae of both species: sensilla basiconica (three subtypes), coeloconica, chaetica (four subtypes), and trichodea. However, sensilla basiconica subtype IV are only present on the antennal surface of S. nigrogilvum. Sensilla trichodea are the most abundant among the four types of sensilla that occur on the antennae of both species. Significant differences in the length of the antennae (scape and flagellomere IX), length of the maxillary palpi (whole and palpal segments I, III, IV and V), and the length and basal width of four sensilla types (trichodea, chaetica, basiconica, and coeloconica) were found. In addition, two types of sensilla were observed on the maxillary palpi: sensilla chaetica (three subtypes) and bulb-shaped sensilla. Differences were observed in the numbers of bulb-shaped sensilla in the sensory vesicles of S. nigrogilvum and S. umphangense. The findings are compared with the sensilla of other insects, and the probable functions of each sensillum type are discussed. The anatomical data on sensory organs derived from this study will help to better understand black fly behavior.

A new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Thailand: Description and phylogenetic relationship with its Thai members.

Simulium (Simulium) thilorsuense sp. nov. is described from females, males, pupae and mature larvae in Thailand. It is placed to the S. striatum species-group, and is distinguished from all the seven species of this species-group recorded from Thailand in the male by the greater number of male upper-eye (large) facets in 20 vertical columns and 19 or 20 horizontal rows, and in the pupa by the combination of the arrangement (2+3+3+2 from dorsal to ventral) and subequal thickness of gill filaments, and tubercles on the frons without secondary projection. It is similar in the female, male and pupa to S. (S.) pingtungense Huang & Takaoka from Taiwan but is distinguished in the larva by the presence of paired protuberances on abdominal segments 1-8 (absence in the latter species). It is similar to S. (S.) grisescens Brunetti from India, S. (S.) perakense Takaoka &Ya'cob from Peninsular Malaysia, and S. (S.) taythienense Takaoka, Sofian-Azirun & Ya'cob from Vietnam by sharing many characteristics including the haired basal portion of the female radius, dark legs of both female and male, male scutum with brassy short hairs, pupal gill with 10 filaments of the almost same thickness, arranged as 2+3+3+2 from dorsal to ventral, and larval abdomen with paired dorsal protuberances, but is distinguished from these three species by the greater number of male upper-eye (large) facets. Our genetic analysis using the fast-evolving nuclear big zinc finger (BZF) gene sequences revealed that this new species has an intraspecific variation ranging from 0.00% to 1.38%, and is separated from six Thai known species of the S. striatum species-group with an interspecific variation of 2.00-4.56%.

A new species of Simulium (Gomphostilbia) (Diptera, Simuliidae) from Thailand, with a key to identify females of 14 species of the Simuliumvaricorne species-group.

Simulium (Gomphostilbia) khelangensesp. nov. is described on the basis of females, collected by a sweeping net in Lampang, Phitsanulok and Chiang Mai Provinces, Thailand. This new species is placed in the S.chumpornense subgroup of the S.varicorne species-group in the subgenus Gomphostilbia Enderlein by having the antenna with eight flagellomeres, pleural membrane bare, and female subcosta lacking hairs. It is similar to S.kuvangkadilokae Pramual & Tangkawanit from Thailand in the same subgroup but is barely distinguished from the latter species by the head width relative to the greatest width of the frons and length of the labrum relative to the clypeus. A genetic analysis using the COI gene sequences similarly shows that S.khelangense sp. nov. is most closely related to S.kuvangkadilokae, with a genetic distance of 1.23-2.81%. A revised key to identify females of 14 species of the S.varicorne species-group is provided.

Morphological description and phylogenetic position of a new cryptic species in the Simulium asakoae species-group, Simulium (Gomphostilbia) mokroense, from Western Thailand.

Adult females and males reared from pupae of Simulium sp. from Tak Province and those of S. chiangdaoense Takaoka & Srisuka from four localities in Chiang Mai and Lampang Provinces, northern Thailand, were molecularly and morphologically compared. Simulium sp. is morphologically almost indistinguishable from S. chiangdaoense except the hair tuft at the base of the radius of the adult female and male, which is composed of yellow and dark hairs (in place of yellow hairs only). Molecular analysis using COI gene sequences shows that S. sp. formed a distinct clade and was separated from S. chiangdaoense by a genetic distance of 1.56-2.44%. Based on the results of morphological and molecular analyzes, S. sp. is described as a new species, S. mokroense, from females, males, pupae and mature larvae. It is also found that S. chiangdaoense is genetically diverse, with five lineages, and is morphologically variable in the number of male upper-eye (large) facets, which are in 13 to 17 vertical columns and 14 to 17 horizontal rows on each side. This is a first case, in which the difference in the color of the wing tuft hairs is a clue leading to the discovery of a new cryptic species close to S. chiangdaoense.

New Insights into Antimalarial Chemopreventive Activity of Antifolates.

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, a thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, has yet to be achieved. Using a transgenic Plasmodium berghei harboring quadruple mutant dhfr from Plasmodium falciparum (Pb::Pfdhfr-4M), we demonstrated that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not to a new class of antifolate designed to overcome the resistance, such as P218. Detailed investigation to pinpoint stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but it is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data show that P218 also inhibited the late hepatic-stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg of body weight, P218 prevented infection from up to 25,000 pyrimethamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates as a chemopreventive therapeutic which could lead to a more efficient and cost-effective treatment regime.

Diversity of nematodes infecting the human-biting black fly species, Simulium nigrogilvum (Diptera: Simuliidae) in central Thailand.

Black flies (Diptera: Simuliidae) are known as vectors of disease agents in humans and livestock, with some species being vectors of Onchocerca volvulus, the filarial nematode that is the causative agent of human onchocerciasis. Nematode infections in adult female black flies have been reported from some areas in northern and western Thailand, but not from other regions of Thailand. In this study, wild-caught adult female black flies from the central region of Thailand were examined for infections with nematodes. Collections of adult females were carried out at Khlong Lan district, Kamphaeng Phet province, central Thailand. A molecular approach, based on the mitochondrial (cox1, 12S rRNA) and nuclear (18S rRNA) genes, was used to identify the species of nematodes recovered from the specimens collected. A total of 911 wild-caught adult black flies were collected. Simulium nigrogilvum was the most abundant species (n = 708), followed by S. doipuiense complex (n = 179), S. chamlongi (n = 11), S. umphangense (n = 10), S. chumpornense (n = 1), S. multistriatum species-group (n = 1), and S. maewongense (n = 1). Nematode infections were detected in nine specimens of S. nigrogilvum, of which two were positive for filarial worms (one worm each, infection rate 0.28%) and seven were positive for non-filarial nematodes (11 worms in total, infection rate 0.99%). The two filarial nematodes (third-stage larvae) were identified molecularly as Onchocerca species type I, while the 11 non-filarial nematodes were classified into ascaridoid (n = 2), tylenchid (n = 6) and mermithid (n = 3) nematodes. The results of this study demonstrated that adult female S. nigrogilvum were parasitized with diverse nematodes (filarial and non-filarial). Detection of the infective larvae of Onchocerca sp. type I in S. nigrogilvum confirms that occurrence of zoonotic onchocerciasis is highly possible in Thailand. Additional in-depth investigation of the morphology, life cycle and host-parasite relationship of nematodes that parasitized this black fly host is still needed.

Community Structure, Biodiversity and Spatiotemporal Distribution of the Black Flies (Diptera: Simuliidae) Using Malaise Traps on the Highest Mountain in Thailand.

Black flies form a group of small blood-sucking insects of medical and veterinary importance. This study aimed to investigate the community structure, biodiversity and spatial and temporal distribution of adult black flies in tropical rain forests, by using malaise traps in Doi Inthanon National Park, northern Thailand. Malaise traps were placed along six elevational gradients (400 m to 2500 m, above sea level) at Doi Inthanon National Park, Chiang Mai province, from December 2013 to November 2014. A total of 9406 adult female black flies belonging to five subgenera-Daviesellum (2%), Gomphostilbia (23%), Montisimulium (11%), Nevermannia (16%) and Simulium (48%)-were collected. Among 44 taxa found, S. tenebrosum complex had the highest relative abundance (11.1%), followed by the S. asakoae species-group (9.6%), the S. striatum species-group (7.7%), S. inthanonense (6.6%), S. doipuiense complex (6.4%), S. chomthongense complex (5.3%), S. chumpornense (5.1%) and S. nigrogilvum (4.1%). Two human-biting species-S. nigrogilvum and species in the S. asakoae species-group-were found in all of the collection sites with 100% species occurrence. Species richness was highest at mid elevation (1400 m), which is represented by 19 black fly species. The peak and lowest seasonal abundance was observed in the rainy and hot season, respectively. Seasonal species richness was highest in the cold season, except for that from elevation sites at 700 m, 1700 m and 2500 m. This study revealed that the malaise trap is effective in providing important data for further monitoring of the effects of environmental changes and conservation planning on the biodiversity of black flies in Doi Inthanon National Park.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Northern Thailand, with its genetic relationship in the S. asakoae species-group.

A new black fly species, Simulium (Gomphostilbia) kiewlomense, is described from females, males, pupae and mature larvae in Thailand. This new species is placed in the S. asakoae species-group and is characterized by having a combination of the elongate female sensory vesicle, widened male hind basitarsus, which is much wider than the hind femur, small pupal terminal hooks, and light greenish larval abdominal segments 1-3. Taxonomic notes are given to separate this new species from other related species. A DNA analysis using the COI gene shows that this new species has two genoforms with 1.21% difference. This is the 28th species of the S. asakoae species-group in Thailand, strengthening the evidence for high species diversity of this species-group.

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus.

Vector control, the most efficient tool to reduce mosquito-borne disease transmission, has been compromised by the rise of insecticide resistance. Recent studies suggest the potential of mosquito-associated microbiota as a source for new biocontrol agents or new insecticidal chemotypes. In this study, we identified a strain of Serratia marcescens that has larvicidal activity against Anopheles dirus, an important malaria vector in Southeast Asia. This bacterium secretes heat-labile larvicidal macromolecules when cultured under static condition at 25°C but not 37°C. Two major protein bands of approximately 55 kDa and 110 kDa were present in spent medium cultured at 25°C but not at 37°C. The Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of these two protein bands identified several proteases and chitinases that were previously reported for insecticidal properties against agricultural insect pests. The treatment with protease and chitinase inhibitors led to a reduction in larvicidal activity, confirming that these two groups of enzymes are responsible for the macromolecule's toxicity. Taken together, our results suggest a potential use of these enzymes in the development of larvicidal agents against Anopheles mosquitoes.

Three new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Thailand, with their phylogenetic relationships.

Three new species of black flies from Thailand, Simulium wangkwaiense, S. tadtonense and S. maeklongkeense, are described based on their adults, pupal exuviae and cocoons. All three new species are assigned to the Simulium (Simulium) striatum species-group, bringing its total number in Thailand to seven. Simulium wangkwaiense sp. nov. is the species formerly called S. quinquestriatum in Thailand. Certain male and pupal morphological characteristics are shown to separate all seven Thai species of this species-group. All of the three new species have been analyzed genetically for their phylogenetic relationships, with three known related species (except for S. thailandicum), by using the fast-evolving nuclear big zinc finger (BZF) gene.

Seasonal Filarial Infections and Their Black Fly Vectors in Chiang Mai Province, Northern Thailand.

The transmission of zoonotic filarial parasites by black flies has so far been reported in the Chiang Mai and Tak provinces, Thailand, and the bites of these infected black flies can cause a rare disease-human zoonotic onchocerciasis. However, species identification of the filarial parasites and their black fly vectors in the Chiang Mai province were previously only based on a morphotaxonomic analysis. In this study, a combined approach of morphotaxonomic and molecular analyses (mitochondrial cox1, 12S rRNA, and nuclear 18S rRNA (SSU HVR-I) genes) was used to clarify the natural filarial infections in female black flies collected by using human and swine baits from two study areas (Ban Lek and Ban Pang Dang) in the Chiang Mai province from March 2018 to January 2019. A total of 805 and 4597 adult females, belonging to seven and nine black fly taxa, were collected from Ban Lek and Ban Pang Dang, respectively. At Ban Lek, four of the 309 adult females of Simulium nigrogilvum were positive for Onchocerca species type I in the hot and rainy seasons. At Ban Pang Dang, five unknown filarial larvae (belonging to the same new species) were detected in Simulium sp. in the S. varicorne species-group and in three species in the S. asakoae species-group in all seasons, and three non-filarial larvae of three different taxa were also found in three females of the S. asakoae species-group. This study is the first to molecularly identify new filarial species and their vector black fly species in Thailand.

Genomic studies on Strongyloides stercoralis in northern and western Thailand.

BACKGROUND: Strongyloidiasis is a soil borne helminthiasis, which in most cases is caused by Strongyloides stercoralis. Human infections with S. fuelleborni fuelleborni and S. fuelleborni kellyi also occur. Although up to 370 million people are currently estimated to be infected with S. stercoralis, this parasite is frequently overlooked. Strongyloides stercoralis is prevalent among humans in Thailand; however, S. fuelleborni fuelleborni has also been reported. Three recent genomic studies of individual S. stercoralis worms found genetically diverse populations of S. stercoralis, with comparably low heterozygosity in Cambodia and Myanmar, and less diverse populations with high heterozygosity in Japan and southern China that presumably reproduce asexually. METHODS: We isolated individual Strongyloides spp. from different localities in northern and western Thailand and determined their nuclear small ribosomal subunit rDNA (18S rDNA, SSU), in particular the hypervariable regions I and IV (HVR-I and HVR-IV), mitochondrial cytochrome c oxidase subunit 1 (cox1) and for a subset whole genome sequences. These sequences were then compared with each other and with published sequences from different geographical locations. RESULTS: All 237 worms isolated from 16 different human hosts were S. stercoralis, no S. fuelleborni was found. All worms had the common S. stercoralis SSU HVR IV haplotype A. Two different SSU HVR I haplotypes (I and II), both previously described in S. stercoralis, were found. No animal heterozygous for the two haplotypes was identified. Among the twelve cox1 haplotypes found, five had not been previously described. Based upon the mitochondrial cox1 and the nuclear whole genome sequences, S. stercoralis in Thailand was phylogenetically intermixed with the samples from other Southeast Asian countries and did not form its own branch. The genomic heterozygosity was even slightly lower than in the samples from the neighboring countries. CONCLUSIONS: In our sample from humans, all Strongyloides spp. were S. stercoralis. The S. stercoralis from northern and western Thailand appear to be part of a diverse, intermixing continental Southeast Asian population. No obvious indication for genetic sub-structuring of S. stercoralis within Thailand or within the Southeast Asian peninsula was detected.

Natural infections with larvae of Onchocerca species type I in the human-biting black fly, Simulium nigrogilvum (Diptera: Simuliidae), in western Thailand.

Zoonotic onchocerciasis is a human infection caused by Onchocerca species of animal origins and transmitted by black fly vectors. The reported incidence of this disease has increased throughout the world. This study aims to clarify the vectorial roles of black fly species in zoonotic filarial transmission in Tak province, western Thailand. The integrated approach of morphological and DNA sequence-based analyses was used to identify species of both wild-caught female black flies and infective filarial larvae found in the infected black flies. All of 494 female black flies captured were identified as Simulium nigrogilvum, through scanning electron microscopy (SEM) and DNA sequence analyses based on the cytochrome c oxidase subunit I (COI) and subunit II (COII), and the fast-evolving nuclear elongation complex protein 1 (ECP1) genes. Four females of S. nigrogilvum harbored one to three third-stage larvae (infective larvae) in their thoraces, with an infection rate of 0.81% (4/494). All infective larvae were similar in morphology and size to one another, being identified as Onchocerca species type I (= O. sp. type A), a bovine filaria, originally reported from Japan, and also as O. sp. found in S. nodosum in Thailand, based on their body lengths and widths being 1,068-1,346 µm long by 25-28 µm wide, and morphological characters. Comparisons of cytochrome c oxidase subunit I (COI) and 12S rRNA sequences of mitochondrial DNA (mtDNA) and phylogenetic analyses with those of previous reports strongly supported that all larvae were O. sp. type I. This report is the first indicating the presence of O. sp. type I in Thailand and its vector being S. nigrogilvum.