Kittipat Aupalee

Black flies are a group of hematophagous insects with high species diversity, driven by a combination of ecological, evolutionary, and life-history factors. By integrating DNA barcoding with traditional morphological method, a new black fly species was discovered. Simulium (Gomphostilbia) wiangpingense sp. nov. is described based on females, males, pupae, and mature larvae collected from Chiang Mai province, Thailand. This new species is assigned to the S. burtoni subgroup of the S. varicorne species-group within the subgenus Gomphostilbia Enderlein, 1921. It is characterized in the female and male adults by the antennae with eight flagellomeres, and pleural membrane bare; in the female by the sensory vesicle elongate, 0.62 to 0.70 times length of the third palpal segment, mandible without teeth on the outer margin, and wing with the subcosta haired; in the male by the sensory vesicle ellipsoidal, 0.42 to 0.43 times length of the third palpal segment; in the pupa by the gill with eight filaments arranged as 3 + 3 + 2 from dorsal to ventral; and in the larva by the long postgenal cleft. Morphologically and genetically, the new species is most closely related to S. burtoni Takaoka & Davies, 1995. Phylogenetic analysis and species delimitation clearly distinguish the new species from its congeners, providing additional support for its recognition as a distinct species. Taxonomic notes for distinguishing the new species from related species within the S. burtoni subgroup from Thailand and other countries, and identification keys to all the 16 species of the S. varicorne species-group are provided.

Although black fly (Diptera: Simuliidae) ecology has been extensively studied in forest ecosystems, knowledge from rural landscapes—where human and livestock interactions are frequent—remains limited. This study investigated the diversity, community structure, and environmental associations of black fly larvae in rural streams of Mae Rim district, Chiang Mai province, northern Thailand. Larvae and pupae collected monthly from three types of study areas were evaluated. Environmental variables were recorded in situ. A total of 3,649 last instar larvae representing 19 species across three subgenera were identified. Dominant species included Simulium nodosum (15.2%), S. chiangmaiense (14.1%), S. wangkwaiense (11.9%), S. asakoae (10.3%), and S. fenestratum (9.5%). Four human-biting species—S. asakoae, S. chamlongi, S. nodosum, and S. doipuiense complex—were identified, with the first three predominantly found in village sites. Species richness and diversity were higher in sites without human intervention (forest without human activity) compared to those in forest with human activity and villages. Canonical Correspondence Analysis (CCA) revealed that conductivity, stream width, water velocity, and salinity were significantly associated with species composition (χ² = 1.53, P < 0.01). Notably, human-biting species were associated with wider, warmer, fast-flowing streams with elevated conductivity—conditions typical of disturbed village sites. These findings underscore the ecological significance of stream conditions in shaping black fly communities and provide critical insights for vector surveillance and control in human-impacted landscapes.

Published studies and records of species of Anopheles that occur in Thailand are reviewed and lists in public databases are revised. ITS2 and COI sequences of specimens from Thailand available in GenBank were analyzed using a phylogenetic method and compared with sequences of species from other countries. Eighty-one species of Anopheles are currently known to occur in Thailand, including the informally recognized An. aconitus species B, An. annularis species A and B, An. jeyporiensis species B, An. tessellatus species A, C, and F, and An. subpictus species C and D. Three new species complexes, the Aconitus, Jeyporiensis, and Karwari Complexes, are recognized.

Utilizing the COI barcoding approach, cryptic diversity has previously been detected within the morphologically recognized Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler, 2008 and S. (G.) sheilae Takaoka & Davies, 1995, of the S. (G.) ceylonicum species-group. Here, an unknown black fly species belonging to the S. ceylonicum species-group from southern Thailand was discovered and described as a new species, S. (G.) sipoense sp. nov. In addition, S. (G.) trangense is herein fully redescribed based on specimens collected from its type locality. Based on an integrative taxonomic approach combining morphological and molecular data, the validity of the newly described S. sipoense sp. nov. and the redescribed S. trangense is confirmed. Comparative morphological characteristics and phylogenetic analysis, inferred from COI sequences, suggest that the new species is conspecific with the species redescribed as S. trangense, using specimens collected from Malaysia, and is morphologically and phylogenetically closely related to S. sheilae, particularly to the specimens from Indonesia. The redescribed S. trangense is genetically highly similar or even identical to the species that was apparently misidentified as S. sheilae from southern and western Thailand, and is morphologically very similar to the new species, from which it is clearly distinguished by the relative length of the female claw tooth, shape of the male ventral plate, and color of the larval body. A detailed information on the morphological characteristics separates the new species, and the redescribed S. trangense from all other known species of the same species-group in Thailand and neighboring countries is provided. Further studies are warranted to clarify the taxonomic status of several cryptic species recognized within the morphologically defined S. trangense and S. sheilae.

Generally, the DNA barcode relying on a short fragment of the cytochrome c oxidase I (COI) gene is a powerful tool for facilitating species discovery and taxonomic resolution in Diptera, including black flies. However, the COI barcode lacks sufficient resolution to identify several species or infer phylogenetic relationships of black flies in the Simulium striatum species-group, whereas the fast-evolving nuclear big zinc finger (BZF) gene has been suggested as a key marker for identifying the species. In this study, a new species of black fly in the S. striatum species-group from Kamphaeng Phet province, central Thailand, was discovered and characterized through an integrated method combining morphological analysis and molecular data based on the BZF gene. The new species, Simulium (Simulium) concitatum sp. nov., was morphologically described for all life stages, excluding the egg. It shares many morphological similarities with other species of the S. striatum species-group, particularly S. thilorsuense Takaoka, Srisuka & Saeung, 2022 described from Tak province, western Thailand. Sequence analysis and phylogeny inferred from the BZF gene further confirmed that S. concitatum sp. nov. is a distinct species of the S. striatum species-group and revealed its close genetic relationship to S. wangkwaiense Takaoka, Srisuka & Saeung, 2020. The morphological differences between the new species and all known species of the S. striatum species-group documented in Thailand and other countries are provided to assist in species identification. Furthermore, this study underscores the BZF gene as an effective genetic marker to differentiate the species.

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.

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.

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.