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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.

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.

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are used as biocontrol agents for insect pests. Survey of indigenous EPNs provides not only the diversity aspects but also the contribution in pest management in local areas. The objective of this study was to survey EPNs in upper northern Thailand. Nine hundred seventy soil samples were obtained from 194 sites in upper northern region of Thailand; of these 60 (6.2%) had EPNs in 2 genera: Steinernema (32 isolates) and Heterorhabditis (28 isolates). Most EPNs were isolated from loam with a soil temperature of 24-38°C, a pH of 1.5-7.0 and a soil moisture content of 0.5-6.8%. Molecular identification based on sequencing of a partial region of an internal transcribed spacer was performed for Heterorhabditis and the 28S rDNA for Steinernema. A BLASTN search of known sequence EPNs revealed 24 isolates of S. websteri and one isolate of S. scarabaei were identified; closely related to S. websteri (accession no. JF503100) and S. scarabaei (accession no. AY172023). The Heterorhabditis species identified were: H. indica (11 isolates), H. gerrardi (2 isolates) and Heterorhabditis sp (8 isolates). Phylogenetic analysis revealed 11 isolates of Heterorhabditis were related to H. indica; 2 isolates were related to Heterorhabditis gerrardi and 8 isolates were closely related to Heterorhabditis sp SGmg3. The study results show the genetic diversity of EPNs and describe a new observation of S. scarabaei and H. gerrardi in Thailand. This finding is new and provides important information for further study on using native EPNs in biological control.

Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin action. There are limited reports identifying such organisms in the National Park of Thailand. Therefore, the objectives of this study were to identify EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand and to evaluate the larvicidal activity of bacteria against Aedes aegypti and Ae. albopictus. A total of 12 EPN isolates belonging to Steinernema and Heterorhabditis were obtained form 940 soil samples between February 2014 and July 2016. EPNs were molecularly identified as S. websteri (10 isolates) and H. baujardi (2 isolates). Symbiotic bacteria were isolated from EPNs and molecularly identified as P. luminescens subsp. akhurstii (13 isolates), X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). For the bioassay, bacterial suspensions were evaluated for toxicity against third to early fourth instar larvae of Aedes spp. The larvae of both Aedes species were orally susceptible to symbiotic bacteria. The highest larval mortality of Ae. aegypti was 99% after exposure to X. stockiae (bNN112.3_TH) at 96 h, and the highest mortality of Ae. albopictus was 98% after exposure to P. luminescens subsp. akhurstii (bNN121.4_TH) at 96 h. In contrast to the control groups (Escherichia coli and distilled water), the mortality rate of both mosquito larvae ranged between 0 and 7% at 72 h. Here, we report the first observation of X. vietnamensis in Thailand. Additionally, we report the first observation of P. luminescens subsp. akhurstii associated with H. baujardi in Thailand. X. stockiae has potential to be a biocontrol agent for mosquitoes. This investigation provides a survey of the basic diversity of EPNs and symbiotic bacteria in the National Park of Thailand, and it is a bacterial resource for further studies of bioactive compounds.

Objective To determine the prevalence of intestinal parasites in children from six schools located in Bang Rakam and Mueang Districts, Phitsanulok Province, Northern Thailand. Methods The study sites were the following six schools: Wat Krab Phuang, Wat Wang Ped, Wat Tha Ko, Wat Sao Hin, Wat Kung Waree and Wat Sakat Namman. Fecal samples were collected from 352 students in the age range 7-15 years old, and examined for intestinal parasites by formalin-ethyl acetate concentration method. Additionally, Scotch tape technique was used to recover Enterobius vermicularis eggs from 576 children. Result It was found that the overall prevalence of intestinal parasites among 352 examined students was 5.4%: Strongyloides stercoralis (2.5%), Entamoeba coli ( E. coli ) (0.6%), Giardia lamblia (0.6%), Enterobius vermicularis (0.6%), hookworm (0.3%), hookworm and E. coli (0.3%), hookworm and Trichuris trichiura (0.3%) and Strongyloides stercoralis and E. coli (0.3%). The overall prevalence of enterobiasis among 576 schoolchildren by Scotch tape technique was 14.1%. The highest infection rate (19.3%) was observed at Wat Sao Hin School. The selective anthelminthic drugs were administered to all positive cases. Conclusions In this study, low prevalence of parasitic infections was demonstrated among schoolchildren in Phitsanulok Province. However, the control and prevention including the transmission of parasites detected should be explained for more concern and attention and the proper hygienic health education should be implemented.

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Entomopathogenic nematodes (EPNs) are used successfully for biological control of subterranean larval pests leading to reduced environmental contamination if chemical control measures are employed. Their diversity and distribution in Thailand are unclear, so the present study sought to obtain a better understanding these EPN populations in the lower northern region of Thailand. We collected 930 soil samples from 186 sites of Kamphaeng Phet, Nakhon Sawan, Phetchabun, Phichit, Phitsanulok, Sukhothai, Tak, Uthai Thani, and Uttaradit Provinces, Thailand from December 2011 to November 2012. Galleria mellonella was used as host for isolating and propagating EPNs. Seventy soil samples (7.5%) yielded EPNs of two genera, Steinernema (3.0%) and Heterorhabditis (4.5%). The majority of the isolated EPNs were found in loam at 26°C-33°C and pH values of 5.0-7.0. Molecular identification from partial 28S rDNA sequences revealed S. websteri, isolated from soil samples from Nakhon Sawan and Uthai Thani. Phylogenetic analysis of these EPNs showed they are closely related to S. websteri JC1032. The identification that S. websteri was the predominant EPN should enable its application for biological control in the local prevailing soil conditions.

International audience; 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.

Sandflies vary in their distributions and role in pathogen transmission. Attempts to record distributions of sandflies in Thailand have faced difficulties due to their high abundance and diversity. We aim to provide an insight into the diversity of sandflies in Thailand by (i) conducting a literature review, and (ii) DNA barcoding sandflies collected from Wihan Cave where eight morphologically characterized species were recorded. DNA barcodes generated for 193 sandflies fell into 13 distinct species clusters under four genera (Chinius, Idiophlebotomus, Phlebotomus and Sergentomyia). Five of these species could be assigned Linnaean species names unambiguously and two others corresponded to characterized morphospecies. Two species represented a complex under the name Sergentomyia barraudi while the remaining four had not been recognized before in any form. The resulting species checklist and DNA barcode library contribute to a growing set of records for sandflies which is useful for monitoring and vector control.

Objective To survey the Angiostrongylus cantonensis ( A. cantonensis ) or the rat lungworm in a rat, definitive host, and in a giant African land snail (Achatina fulica), the intermediate host, in Phitsanulok, Thailand. Methods Rats and giant African land snails were captured from Tha Pho sub-district, Phitsanulok, Thailand. Rats were killed and examined for adult A. cantonensis . The artificial digestion method following Baermann technique were used for isolation third stage larvae of A. cantonensis . Results Sixty-two rats were captured and they were identified as Rattus argentiventer, Rattus rattus ( R. rattus ), Bandicota savilei , and Bandicota indica but only one animal ( R. rattus ) of 62 rats (1.61%) was positive with adult worm of A. cantonensis . The third stage larvae of A. cantonensis were examined on 307 Angiostrongylus fulica snails. It was found that the overall infection rate was 12.38% (38 infected out of 307 Achatina snails). Conclusions This study demonstrates that A. cantonensis is available in the natural hosts of Phitsanulok. This suggests that the transmissions of this parasite to human may occur in this region.