Jiraporn Ruangsittichai

Subtype Distribution of Blastocystis in Thai-Myanmar Border, Thailand

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.

Interspecific and sexual shape variation in the filariasis vectors Mansonia dives and Ma. bonneae.

In the South of Thailand, six Mansonia species are recorded as filariasis vectors, among which Ma. bonneae and Ma. dives. These two species are distributed in the same breeding place, mainly the swamp forest, but appear to be of problematic identification using traditional morphological characters. Because of the risk of wrong identification during epidemiological or biological studies, complementary techniques are needed to distinguish the two species. We used on the same field collected specimens both genetic (DNA barcoding) and phenetic (geometric morphometrics) techniques. Both methods converged to identify two separate entities in accordance with morphological differences and geographic origins. Shape divergence between species was more pronounced in males than in females. Notably, the amount of within species sexual shape dimorphism was much larger than shape divergence as recorded between species. In spite of these two species of Mansonia being evolutionary very close, simple DNA barcoding was resolutive. Geometric morphometrics, because it is a fast and low-cost procedure, appeared as an interesting complement to modern diagnostic techniques applied in medical entomology. It also was able to provide information relevant to the ecology of the two species.

A molecular phylogeny of Asian species of the genus Metagonimus (Digenea)—small intestinal flukes—based on representative Japanese populations

Metagonimus Katsurada, 1912 is a genus of small intestinal parasites. The genus comprises eight species, primarily from far-eastern Asia, with two exceptions reported from Europe. Metagonimus yokogawai, the most widespread species, is the main agent responsible for the intestinal disease, metagonimiasis, in Japan and some other East Asian countries. On the basis of the ratio of the size of the ventral and oral suckers, Metagonimus has traditionally been morphologically divided into two groups; however, the genus has not been extensively studied using molecular data. To reveal phylogenetic relationships within Metagonimus based on molecular data, we analyzed six of the seven species present in Asia using samples collected in central Japan. Maximum likelihood and Bayesian analyses of a combined 28S ribosomal DNA (rDNA), internal transcribed spacer 2 (ITS2), and mitochondrial cox1 gene sequence dataset separated the six species into two well-supported clades. One clade comprised M. yokogawai, M. takahashii, M. miyatai, and M. hakubaensis, whereas the other consisted of M. otsurui and M. katsuradai. Genetic distances calculated from 28S rDNA and ITS2 nucleotide sequences and a comparison of the predicted amino acid sequences of cox1 gene suggested that M. otsurui and M. katsuradai may have diverged recently. None of the four main morphological characters used to delimit species of Metagonimus (i.e., sucker ratio, positions of the uterus and testes, and distribution of vitelline follicles) was consistent with the distribution of species in the molecular tree.

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

DNA barcoding and wing morphometrics to distinguish three Aedes vectors in Thailand

Aedes aegypti (Diptera: Culicidae) (L.), Ae. albopictus (Skuse), and Ae. scutellaris (Walker) are important mosquito vectors of dengue and chikungunya viruses. They are morphologically similar and sympatric in some parts of their distribution; therefore, there is a risk of incorrect morphological identification. Any confusion could have a negative impact on epidemiological studies or control strategies. Therefore, we explored two modern tools to supplement current morphological identification: DNA barcoding and geometric morphometric analyses. Field larvae were reared to adults and carefully classified based on morphological traits. The genetic analysis was based on the 658bp each of 30COI sequences. Some Culex spp., Mansonia bonneae, were included as outgroups, and inclusion of a few other Aedes spp. facilitated phylogenetic inference of the relationship between Ae. albopictus and Ae. scutellaris. The two species were separated by an average interspecific divergence of 0.123 (0.119-0.127). Morphometric examination included landmark- (392 specimens) and outline-based (317 specimens) techniques. The shape of the wing showed different discriminating power based on sex and digitizing technique. This is the first time that Ae. scutellaris and Ae. albopictus have been compared using these two techniques. We confirm that these morphologically close species are valid, and that geometric morphometrics can considerably increase the reliability of morphological identification.

Species-Specific Primers Designed from RAPD Products for Bithynia funiculata, the First Intermediate Host of Liver Fluke, Opisthorchis viverrini, in North Thailand

Abstract: Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

Landmark and outline-based geometric morphometrics analysis of three Stomoxys flies (Diptera: Muscidae).

Adult flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), especially S. pullus Austen, 1909, S. uruma Shinonaga et Kano, 1966 and S. indicus Picard, 1908, are morphologically similar and sometimes difficult to distinguish when using external morphological characteristics. These species may act as vectors and/or potential vectors of many pathogens (virus, bacteria and protozoa). Their correct identification is important to target the vectors involved in the transmission of the pathogens and also helps in the fly control program.The aim of the present study was to distinguish three species which are difficult to separate using traditional diagnostic characters for species of Stomoxys such as colour patterns and body proportions. Modern morphometrics, both landmark and outline-based, was used to access wing geometry of S. pullus, S. uruma and S. indicus. A total of 198 and 190 wing pictures were analysed for landmark- and outline-based approaches, respectively. Wing shape was able to separate species and sexes of the three Stomoxys flies with highly significant difference of Mahalanobis distances. The cross-validated classification scores ranged from 76% to 100% for landmark and 77% to 96% for outline-based morphometrics. The geometry of wing features appears to be a very useful, low-cost tool to distinguish among the vectors S. pullus, S. uruma and S. indicus.

Species identification of horse flies (Diptera: Tabanidae) in Thailand using DNA barcoding

Horse flies (Diptera: Tabanidae) are of medical and veterinary importance because they are known to transmit pathogens. Approximately 80 species of horse flies have been reported in Thailand. Monitoring the distribution of horse fly species is important to control the spread of diseases transmitted by them. Currently, the species identification of horse flies is based on their morphology; this requires considerable skills and taxonomic expertise, and it may be difficult to identify morphologically similar species. DNA-based identification methods are increasingly being developed for rapid and accurate identification of various insect species. In this study, we used mitochondrial cytochrome oxidase subunit I (COI) for species identification of horse flies in Thailand. A 658 bp fragment of COI was amplified from 145 adult horse flies belonging to 48 morphologically distinct species and sequenced. Sequence analysis revealed an intraspecific divergence of 0.0%-4.4% and an interspecific divergence of 0.0%-16.2%. Our results showed that COI barcodes were effective in discriminating a majority of horse flies in Thailand on the basis of the barcoding gap and phylogenetic analyses. However, COI barcodes were unable to distinguish among members of the Tabanus striatus complex and some species within the T. ceylonicus group.

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.