Distribution of Phlebotomine Sandflies in the Cave Area of Satun Province, Thailand.

Leishmaniasis, a sandfly-transmitted protozoan infection, is a neglected health threat in Thailand and the information on its vector is scarce. This study aimed to identify sandfly distribution, abundance, and environmental conditions of natural breeding sites in the cave areas of Satun Province, where previous cases of leishmaniasis were reported. Sandflies were collected during a six-month period using CDC light traps and modified emergence traps. Species distribution, relative abundance, and environmental conditions of potential breeding sites were determined. Our survey of 12,790 sandflies found the highest female abundance in April-May. We identified six known species, the most prevalent being Sergentomyia anodontis. We also found S. barraudi, a potential Leishmania spp. vector, distributing in this area. Most male sandflies had partially rotated genitalia, indicating the breeding site proximity to our trap locations. Potential resting/breeding sites were discovered outside the cave during February-March, and inside during May-June. The environmental parameters showed warm climate, moderate humidity, moderately alkaline pH, moderate-to-high macronutrients, and low-to-high organic matters. In summary, our study provided the spatiotemporal distribution and environmental condition of sandfly potential breeding sites in the cave areas of Satun Province. This data may contribute to more effective vector surveillance programs in the future.

Geometric morphometrics of the scutum for differentiation of trombiculid mites within the genus Walchia (Acariformes: Prostigmata: Trombiculidae), a probable vector of scrub typhus.

The vectors of scrub typhus are the larval stage of trombiculid mites, termed "chiggers". These vectors are very small - the larvae are approximately 0.2 mm in size - and therefore their morphological identification is difficult. Trombiculid mites are widely distributed across Asia and they can be identified at the genus level by the shape, size and setae/sensilla distribution of their dorsal chitin plate (scutum = shield), while morphological identification at the species level requires more mite characteristics. We recently developed a methodology to ascertain paired matched genotype and morphotype of individual chiggers, based on autofluorescence and brightfield microscopy with subsequent molecular identification using the COI gene (approximately 640bp length). However, based on 20 chigger specimens characterised by paired genotypic and morphological data consisting of the four species [Walchia ewingi with 2 subspecies]: Walchia ewingi lupella (n = 9), W. ewingi ewingi (n = 2), W. alpestris (n = 2), W. kritochaeta (n = 5) and W. minuscuta (n = 2) we found evidence of genetic polymorphism and morphological plasticity within the genus Walchia. The phylogenetic inference of the intra-genus relationships within the Walchia spp., based on COI gene (Blankaartia spp. served as outgroup), delineated the five included species by an average interspecific divergence of mean distance 0.218 (0.126 - 0.323). We therefore applied landmark-based and outline-based geometric morphometric (GM) approaches to differentiate Walchia species using scutum measurements. A total of 261 scutum images of Walchia spp. were examined by landmark-based GM (140 chigger specimens) and outline-based GM (121 specimens) techniques. All Walchia spp. showed significant differences in scutum size and shape. W. minuscuta showed the smallest mean scutum size in both techniques. The largest scutum was found in W. ewingi lupella and W. ewingi ewingi by landmark-based and outline-based GM analysis, respectively. The scutum shapes of W. alpestris and W. minuscuta were clearly distinguished from the other species. Cross-validated classification scores were different depending on species and digitizing techniques and landmark-based GM showed better scores than outline-based GM. We conclude that the morphologically closely-related trombiculid mite species can be further differentiated by their scutum features alone, using GM approaches. This technique is a promising tool for the much-needed characterization studies of chiggers and needs evaluation using matched morphometric and genotyping data for other genera of trombiculids.

Species composition and nocturnal activity of phlebotomine sand flies (Diptera: Psychodidae) inhabiting a limestone cave in Thailand.

We investigated the nocturnal activity of cave-dwelling sand flies at different time intervals and determined their species composition and seasonal variation. Sand flies were captured on one night each month using CDC light traps from 18:00-06:00 with the collecting bag being changed every two h between February, 2010 and January, 2011. A total of 18,709 individuals, including 10,740 males and 7,969 females, was collected. The overall ratio between male and female specimens was 1:0.74. The collected specimens included 14 species from four genera, Chinius, Idiophlebotomus, Phlebotomus, and Sergentomyia. Sergentomyia phadangensis was the most abundant species (comprising 31.9% of the collected individuals), followed by Se. anodontis (22.8%) and Ph. mascomai (18.2%). The highest number of specimens was collected in July (15.6%), followed by May (15.5%) with the peak of collection recorded at the time interval of 00:01-02:00, followed by 22:01-00:00. However, there were no significant differences observed among time intervals of sand fly collections (p=0.154). Observations of the nocturnal activity of male and female sand flies throughout the night suggest that phlebotomine sand flies show the greatest activity level after midnight.

Diversity Of Mosquito Species Ovipositing In Different Zones of Light Intensity Within Limestone Caves In Thailand.

Climate change and human activity affect the geographical and annual distribution and population abundance of mosquitoes. As natural habitats are reduced, it is hypothesized that mosquitoes may seek refuge in more stable environments such as cave habitats. Therefore, we explored the species diversity of mosquitoes exploiting cave habitats in Thailand. Ten species belonging to 4 genera were collected, of which none were considered to be true cave-dwelling species (Troglobiont). The known cavernicolous species, Aedes cavaticus, was observed to oviposit outside of the cave and therefore should be categorized as a subtroglophilic species. Other species were also oviposited inside the cave but should be regarded as trogloxenic species. There was no clear association between environmental factors and mosquito abundance inside the 4 limestone caves, except for Ae. cavaticus, which was positively correlated with rainfall. This study indicates that different biotic or abiotic factors may be involved in mosquito oviposition site selection inside caves.

Wing geometry of Phlebotomus stantoni and Sergentomyia hodgsoni from different geographical locations in Thailand.

Geographic populations of the two main sandflies genera present in Thailand were studied for species and population identification. Size and shape of Phlebotomus stantoni and Sergentomyia hodgsoni from different island and mainland locations were examined by landmark-based geometric morphometrics. Intraspecific and interspecific wing comparison was carried out based on 12 anatomical landmarks. The wing centroid size of P. stantoni was generally larger than that of S. hodgsoni. Within both species, wings from the continent were significantly larger than those from island populations. Size variation could be significant between geographic locations, but could also overlap between genera. The wing venation geometry showed non-overlapping differences between two species. The within-species variation of geometric shape between different geographical locations was highly significant, but it could not interfere with the interspecies difference. The lack of species overlapping in shape, and the high discrimination between geographic populations, make geometric shape a promising character for future taxonomic and epidemiological studies.

IDENTIFICATION OF BAT ECTOPARASITE LEPTOCIMEX INORDINATUS FROM BAT-DWELLING CAVE, KANCHANABURI PROVINCE, THAILAND.

Bat bugs are blood-feeding insects of bats or warm blooded animals and humans. Since 2011, Leptocimex spp (Heteroptera: Cimicidae) has been reported in Thailand. However, microscopic examination of Leptocimex spp is complicated, especially when the entire body of the specimen is not available. To confirm the phenotypic identification of L. inordinatus from a limestone bat cave in Thailand, partial fragments of mitochondrial cytochrome c oxidase subunit 1 (COI) gene and 16S mitochondrial ribosomal DNA were PCR amplified and sequenced, which revealed 97% sequence identity with Cimicidae family members, being most similar to Cacodminae gen. sp. and C. vicinus, both bat bugs. Phylogenetic tree construction showed that L. inordinatus has a separate genetic lineage from that of with human bed bugs (Cimex hemipterus or C. lectularius), swallow bugs and other tick species. The presence of L. inordinatus in a bat-dwelling cave frequented by humans presents a potential public health problem requiring attention in particular regarding the possibility of zoonotic transmission of pathogens.

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.

Description of Sergentomyia phadangensis n. sp. (Diptera, Psychodidae) of Thailand.

BACKGROUND: Since 1996, there are emerging autochthonous cases of leishmaniasis in Thailand due to Leishmania "siamensis" and to L. martiniquensis explaining a recent interest for the sand fly fauna where Sergentomyia gemmea and Se. barraudi have been considered possible vectors in the country. METHODS: Field studies were undertaken in a cave of Phitsanulok Province, Thailand. Phlebotomine sandflies have been studied morphologically and some have been processed for molecular biology (sequencing of cytB rDNA). RESULTS: A new species of sand fly, belonging to the genus Sergentomyia: Se. phadangensis n. sp., is described. The association of the male and female is supported by the homology of the sequences of cytochrome b rDNA. CONCLUSIONS: The description of a new species in Thailand is of importance in view of the existence of autochthonous leishmaniases.

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.

Phlebotomine sand flies of edible-nest swiftlet cave of Lang Ga Jiew Island, Chumphon province, Thailand.

The present study reported for the first time phlebotomine sandfly species inhabiting edible-nest swiftlet cave of the isolated island, based on field collections made during June 2010-May 2011. The insect diversity was relatively lower to that of mainland caves. All species, Phlebotomus stantoni, Sergentomyia anodontis, Sergentomyia bailyi, Sergentomyia gemmea, Sergentomyia hodgsoni and Sergentomyia punjabensis were either endemic island species or native elsewhere in Thailand. Sergentomyia hodgsoni was the most prevalent species accounted for 94.7% and classified as a troglophile species. Seasonal pattern of the phlebotomine abundance and some aspects of their population characteristics were described and discussed. Two ectoparasites, Ornithodorus and Paracimex sp. were also incidentally collected from the swiftlet cave.

CORRELATION OF HOST SPECIFICITY, ENVIRONMENTAL FACTORS AND ORIENTAL RAT FLEA ABUNDANCE.

Fleas are the vectors of many communicable diseases that are normally found in oriental rats. Climate and environmental changes influence the habitat and migration patterns of vectors. In this study, the oriental rat flea abundance, represented as total flea index, was determined in correlation to host specificity and various environmental factors. The number of hosts and fleas calculated from 3 specific habitats (shipping area, decayed area, and market area) from July 2010 to June 2011. The results showed that the common hosts in the shipping area and decayed area were Rattus rattus and R. exulans, with the total flea indexes of 3.36 and 1.58. R. norvegicus was the most common host identified in the market area. Fleas were virtually absent in rat hosts collected from the market area. Both the density of reservoir hosts and the total flea index were positively correlated with the mean annual rainfall and temperature. These data could be useful for control of rat populations in each specific habitat.

Coverage-dependent effect of insecticide-treated curtains for dengue control in Thailand.

Evidence on the effectiveness of insecticide-treated curtains (ITCs) for reducing densities of Aedes mosquitoes, the principal vectors of dengue, is scarce. In Laem Chabang southeast of Bangkok, Thailand, the Breteau Index (BI) (number of positive containers/100 houses) was 45 in October 2006. In March 2007, we distributed long-lasting ITCs in 22 clusters (2,032 houses) and selected 66 control clusters (661 houses). Routine control activities continued in all clusters. Six months after distribution, the BI was 25.8 and 77.6 in intervention and control areas, respectively (P < 0.001). Eighteen months after distribution, the BI was 21.8 and 23.8, respectively (P = 0.28). The average number of ITCs/house at cluster level was associated with the BI (P < 0.01) after six months, when 70.5% of households still used ITCs, but not at 18 months, when ITC coverage had decreased to 33.2%. Deployment of ITCs can result in considerable reductions in Aedes infestation levels, but the effect is coverage dependent.

A cluster-randomized trial of insecticide-treated curtains for dengue vector control in Thailand.

The efficacy of insecticide-treated window curtains (ITCs) for dengue vector control was evaluated in Thailand in a cluster-randomized controlled trial. A total of 2,037 houses in 26 clusters was randomized to receive the intervention or act as control (no treatment). Entomological surveys measured Aedes infestations (Breteau index, house index, container index, and pupae per person index) and oviposition indices (mean numbers of eggs laid in oviposition traps) immediately before and after intervention, and at 3-month intervals over 12 months. There were no consistent statistically significant differences in entomological indices between intervention and control clusters, although oviposition indices were lower (P < 0.01) in ITC clusters during the wet season. It is possible that the open housing structures in the study reduced the likelihood of mosquitoes making contact with ITCs. ITCs deployed in a region where this house design is common may be unsuitable for dengue vector control.

Literature review of parasitoids of filth flies in Thailand: a list of species with brief notes on bionomics of common species.

We reviewed the literature for surveys of parasitoid of filth flies in Thailand. We found 5 families, with 9 genera and 14 species identified in Thailand. We describe the ecological niches and biology of common species, including Spalangia cameroni, S. endius, S. nigroaenea and Pachycrepoideus vindemmiae.

Biology of Culex sitiens, a predominant mosquito in Phang Nga, Thailand after a tsunami.

A tsunami affected area in Phang Nga province, Thailand was explored randomly as some freshwater sites had changed into brackish-water sites. A survey of four areas found Culex sitiens to be the most dominant mosquito species.This mosquito prefers to breed in putrefied water with garbage and it was found in almost every stagnant, brackish-water site in full sunlight. The larval density was more than 300 larvae/dip/250 ml water. Its biting cycle, determined by human landing catch, was nocturnal, with a single peak at 19.00-20.00 hr. The maximum rate was 108 mosquitoes per person/hour. The biology of the mosquito was studied by colonization in natural water under laboratory conditions. The mean number of eggs per raft was 158.1 ± 31.7, hatchability 96.6 ± 4.1%, development from 1st instar larvae to adult was 8.8-11.7 days, and longevity of adult males was 7.3-41.3 days and females 11.0-52.7 days. The ratio of adult males to adult females was 1:1.1 ± 0.2.

Cloning of a trypsin-like serine protease and expression patterns during Plasmodium falciparum invasion in the mosquito, Anopheles dirus (Peyton and Harrison).

Understanding specific gene regulation during responses to malaria infection is key to dissecting the mosquito defense mechanisms and host/parasite interactions. A full-length serine protease cDNA was isolated from the fat body of female Anopheles dirus, a major malaria vector in Thailand. The predicted amino acid sequence of SERF4 identifies it as a member of the serine protease family containing a single trypsin-like protease domain. Digestive trypsins function in the female mosquito midgut and are inducible in two phases in this tissue upon blood intake. However, the gene was highly upregulated in the midgut at day 3 postinfection by Plasmodium falciparum. In situ hybridization confirmed that SERF4 transcripts were located in the midgut epithelial cells rather than hemocytes or other tissues associated with the midgut. SERF4 was also strongly downregulated in the whole insects at day 16 after infection in comparison with the blood-fed control. Changes in the expression of the SERF4 gene in response to infection with this human malaria parasite suggest a role in parasite-specific innate immunity.

Field-collected permethrin-resistant Aedes aegypti from central Thailand contain point mutations in the domain IIS6 of the sodium channel gene (KDR).

One of the mechanisms responsible for pyrethroid resistance in mosquitoes is mutations in domain IIS6 of voltage-gated sodium channel gene (kdr). Aedes aegypti larvae were collected from the central provinces of Thailand (Bangkok, Prachin Buri and Ratchaburi) and colonized until they became adults. Partial fragment of kdr of permethrin-resistant mosquitoes were amplified by RT-PCR and sequenced. Among the four nucleotide mutations detected, two mutations resulted in two amino acid substitutions, S(TCC) 989 P(CCC) and V(GTA)1016 G(GGA). Among 94 permethrin-resistant mosquitoes, the SS genotype (SS/VV) was found to predominate (n = 74), followed by SR (SP/VG) (n = 15) and RR (PP/ GG) genotypes (n = 5), with the resistant allele frequency ranging from 0.03 to 0.17. As pyrethroid insecticides are currently being advocated for use in Thailand, investigations of pyrethroid resistance in other regions of the country are needed to prevent potential cross-resistance among different types of insecticides.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

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.