DNA Barcoding of Culicoides Latreille (Diptera: Ceratopogonidae) From Thailand Reveals Taxonomic Inconsistencies and Novel Diversity Among Reported Sequences.

Recent focus on Culicoides species diversity in Thailand was prompted by a need to identify vectors responsible for the transmission of African Horse Sickness in that country. To assist rapid genetic identification of species, we sampled mitochondrial cytochrome c oxidase subunit I (COI) DNA barcodes (N = 78) from 40 species of Culicoides biting midge from Thailand, including 17 species for which DNA barcodes were previously unavailable. The DNA barcodes were assigned to 39 Barcode Identification Numbers (BINs) representing terminal genetic clusters at the Barcode of Life Data systems (BOLD). BINs assisted with comparisons to published conspecific DNA barcodes and allowed partial barcodes obtained from seven specimens to be associated with BINs by their similarity. Some taxonomic issues were revealed and attributed to the possible misidentification of earlier reported specimens as well as a potential synonymy of C. elbeli Wirth & Hubert and C. menglaensis Chu & Liu. Comparison with published BINs also revealed genetic evidence of divergent population processes and or potentially cryptic species in 16 described taxa, flagged by their high levels of COI sequence difference among conspecifics. We recommend the BOLD BIN system to researchers preparing DNA barcodes of vouchered species for public release. This will alert them to taxonomic incongruencies between their records and publicly released DNA barcodes, and also flag genetically deep and potentially novel diversity in described species.

Phlebotomine Sand Flies in Southern Thailand: Entomological Survey, Identification of Blood Meals and Molecular Detection of Trypanosoma spp.

An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

DNA barcode identification and molecular detection of bluetongue virus in Culicoides biting midges (Diptera: Ceratopogonidae) from western Thailand.

Biting midges of the genus Culicoides Latreille are biological vectors of bluetongue virus (BTV), a member of family Reoviridae, genus Orbivirus. About 30 species of Culicoides have been identified as competent BTV vectors worldwide. Even though high seroprevalence of BTV has been reported among livestock ruminants from western Thailand, the Culicoides species which contribute to BTV transmission remain unclear. In the present study, Culicoides were collected from eight sampling sites, located in two BTV prevalent provinces in western Thailand. Adult Culicoides were identified using wing morphology and cytochrome c oxidase subunit I (COI) mtDNA molecular marker. A total of 9,677 Culicoides specimens belonging to 7 subgenera, 3 species groups, and 23 species were identified. After comparing sequencing results with available data from GenBank, COI sequences of five species were reported for the first time from Thailand. The most abundant potential BTV vector species collected were C. peregrinus, followed by C. orientalis, C. imicola, C. oxystoma, and C. fulvus. Out of 72 Culicoides pools, 9 pools (4 from C. orientalis, 2 from C. imicola, 2 from C. oxystoma, and 1 from C. fulvus) were positive by BTV RT-PCR analyses. These results are new to Culicoides BTV vector knowledge in Thailand and will contribute to further BTV studies in this particular region.

Culicoides Latreille biting midges (Diptera: Ceratopogonidae) of the Dongzhaigang mangrove forest, Hainan Province, China.

The biting midge fauna of Dongzhaigang Mangrove Forest, Hainan Province, China was sampled on 14 October 2015 using three methods: a pan light trap operated from dusk until dawn the following morning and sweep net and human landing collections performed between 16:15-17:15 hr. Eight species, including two new records for China, Culicoides palawanensis and C. niphanae, and one new record for Hainan, C. circumbasalis, were collected. A key to assist with identification of specimens of these species is provided. DNA barcodes supported the morphological identification of some of these species and identified the potential presence of cryptic species and/or deep population structure in others. The newly recorded species were morphologically similar to species previously reported from Hainan, highlighting the need for further investigation into the taxonomy of biting midges in this region. Species composition and abundance varied considerably between the three collection techniques suggesting that multiple techniques likely provide a more comprehensive sample of biting midge fauna.

Ten species of Culicoides Latreille (Diptera: Ceratopogonidae) newly recorded from Thailand.

A survey of biting midges in animal sheds, mangroves and beaches along the Andaman coastal region in southern Thailand between April 2012 and May 2013 collected 10 species of Culicoides which were not previously known from Thailand. These new records are C. arenicola, C. flavipunctatus, C. hui, C. kinari, C. kusaiensis, C. parabubalus, C. quatei, C. spiculae, C. pseudocordiger and C. tamada. An updated checklist of species of Culicoides reported from Thailand is provided.

Integrative taxonomy to investigate species boundaries within Culicoides (Diptera: Ceratopogonidae): a case study using subgenus Avaritia from Australasia and Eastern Asia.

In this study, species boundaries were examined for 15 described and 2 undescribed species within the economically important Culicoides subg. Avaritia Fox from Australasia and Eastern Asia. We used an integrative taxonomic approach incorporating DNA barcoding, nuclear gene sequencing, and retrospective morphological analyses. Some arbovirus vector species such as Culicoides fulvus Sen and Das Gupta and Culicoides wadai Kitaoka were genetically and morphologically uniform across sampled distributions, but others including Culicoides actoni Smith and Culicoides brevipalpis Delfinado contained 2 or more genetically independent populations of 'cryptic species' that in some cases were sympatric. Some of these 'cryptic species' exhibited consistent morphological differences, while differences are yet to be found for others species. Additionally, an undescribed species, C. Avaritia sp. No. 3, was found to be synonymous with C. fulvus. These results refine our understanding of the distribution of individual species of C. subg. Avaritia and demonstrate that species descriptions and distribution records need revision for part of the Culicoides fauna. Furthermore, because vector competence studies for most of these species are based entirely on Australian populations, the competence of the putative cryptic species identified elsewhere will require independent assessment. Finally, integrative taxonomic assessment requires genetic and morphological assessment of material from the type localities in order to clarify the status and distribution of species, especially for clades containing cryptic species. International collaboration is needed to facilitate this research.