Population genetic structure of Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand and its infection with trematode cercariae.

Indoplanorbis exustus is a freshwater gastropod belonging to the family Planorbidae. This snail is widely distributed across the tropics and plays an important role as the intermediate host for trematodes. However, relatively little is understood regarding the genetic relationship between I. exustus and trematodes. The goals of this study were to investigate the current transmission status of trematode cercariae in I. exustus in Thailand and to examine the genetic diversity, genetic structure, and demographic history of I. exustus. We collected 575 I. exustus from 21 provinces across six regions of Thailand and investigated cercarial infections by using the shedding method. I. exustus from two provinces were infected with cercarial trematodes, and two types of cercarial stages were molecularly identified as furcocercous cercaria and xiphidiocercariae. Phylogenetic tree analysis based on 28S rDNA and ITS2 sequences demonstrated that furcocercous cercaria and xiphidiocercariae were closely clustered with a clade of Euclinostomum sp. and Xiphidiocercariae sp., respectively. Phylogenetic and network analyses of I. exustus haplotypes based on the COI, 16S rDNA, and ITS1 genes demonstrated four main clades. Only snails in clade A were distributed in all regions of Thailand and harbored trematode cercariae. The level of genetic diversity of I. exustus was relatively high, but most populations were not genetically different, thus suggesting the appearance of gene flow within the I. exustus populations. Overall, the haplotype network was star-shaped, thus suggesting the recent demographic expansion of populations. This result was also supported by the unimodal mode of the mismatch distribution graph and the large negative values of the neutrality tests. Therefore, the I. exustus snail was likely another freshwater snail of the invasive species in Thailand. This information will aid in monitoring the spread of the parasitic trematodes carried by I. exustus from different populations.

Larvicidal activity of Photorhabdus and Xenorhabdus bacteria isolated from insect parasitic nematodes against Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus are important vectors for several arboviruses such as the dengue virus. The chemical control of Aedes spp., which is usually implemented, affects both humans and the environment. The biological control of Aedes spp. with entomopathogenic bacteria such as Photorhabdus and Xenorhabdus may be an alternative method that can overcome such issues. This study aimed to isolate and identify Photorhabdus and Xenorhabdus bacteria from entomopathogenic nematodes (EPNs) collected in Thailand and evaluate their larvicidal properties in controlling A. aegypti and A. albopictus. Colony morphology and recA sequencing of the 118 symbiotic isolated bacteria indicated that most were P. luminescens subsp. akhurstii and X. stockiae with minor prevalence of P. luminescens subsp. hainanensis, P. asymbiotica subsp. australis, X. indica, X. griffiniae, X. japonica, X. thuongxuanensis, and X. eapokensis. The larvicidal bioassay with the third- and fourth-instar mosquito larvae suggested that a whole-cell suspension of X. griffiniae (bMSN3.3_TH) had the highest efficiency in eradicating A. aegypti and A. albopictus, with 90 ± 3.71% and 81 ± 2.13% mortality, respectively, after 96 h exposure. In contrast, 1% of ethyl acetate extracted from X. indica (bSNK8.5_TH) showed reduced mortality for A. aegypti of only 50 ± 3.66% after 96 h exposure. The results indicate that both X. griffiniae (bMSN3.3_TH) and X. indica (bSNK8.5_TH) could be used as biocontrol agents against Aedes larvae.

Population Genetics Analysis of a Pomacea Snail (Gastropoda: Ampullariidae) in Thailand and its Low Infection by Angiostrongylus cantonensis.

Pomacea is a freshwater snail in family Ampullariidae that is native to South and Central America. This snail is among the more important intermediate hosts for Angiostrongylus cantonensis and agricultural pests. Herein, we investigated the prevalence of A. cantonensis larvae and the genetic diversity of Pomacea samples collected across Thailand based on mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. The larval-infection rate was 1.7% in Pomacea canaliculata specimens collected from the Uttaradit Province of northern Thailand. We randomly selected specimens of P. canaliculata and P. maculata for genetic analysis. We analyzed 244 COI sequences, including 49 sequences from samples collected from Thailand and a publicly accessible database of snails in their native and non-native ranges. A maximum-likelihood tree of P. canaliculata and P. maculata revealed two main clades. The genetic diversity analysis identified seven P. canaliculata haplotypes and six P. maculata haplotypes, and showed genetic differences between the populations of P. canaliculata and P. maculata. The haplotype networks of P. canaliculata and P. maculata populations in Thailand are similar to those of populations in multiple countries, indicating that this species spread widely to many parts of the world.

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.

Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus.

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.

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective:To evaluate the efficacy of symbiotic bacteria,Xenorhabdus indica,Xenorhabdus stockiae,Photorhabdus luminescens subsp,akhurstii and Photorhabdus luminescens subsp.hainanensis as a larvicide against Aedes aegypti and Aedes albopictus.Methods:Larvae (L3-L4) of Aedes aegyptiand Aedes albopictus were given 2 mL of a suspension 107-108 CFU/mL of each symbiotic bacterium.Distilled water and Escherichia coli ATCC· 25922 were used as the control.The morality rate of the larval mosquitoes was observed at 24,48,72 and 96 h.The experiment was performed in triplicates.Results:The larvae of both Aedes species started to die at 24 h exposure.Aedes aegypti showed the highest mortality rate (87％-99％),96 h after exposure to Xenorhabdus stockiae (bNBP22.2_TH).The mortality rate of Aedes albopictus was between 82％ and 96％ at 96 h after exposure to Xenorhabdus indica (bKK26.2_TH).Low effectiveness of distilled water and Escherichia coliATCC· 25922 were observed in both Aedes larvae,with a mortality rate of 2％ to 12％.Conclusions:The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp.Xenorhabdus stockiae and Xenorhabdusindica may be an alternative agent for control Aedes spp.This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

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.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

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.

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.

PRELIMINARY SURVEY OF ENTOMOPATHOGENIC NEMATODES IN UPPER NORTHERN THAILAND.

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.

Unexpected diversity of sandflies (Diptera: Psychodidae) in tourist caves in Northern Thailand.

Certain species of Phlebotomine sandflies (Diptera: Psychodidae) are vectors of the protozoa which causes leishmaniasis. Sandflies are found breeding in enclosed places like caves. Thailand is a popular tourist destination, including for ecotourism activities like caving, which increases the risk of contact between tourists and sandflies. Surveillance of sandflies is important for monitoring this risk but identification of species based on morphology is challenged by phenotypic plasticity and cryptic diversity. DNA barcodes have been used for the identification of sandflies in Thailand. We collected sandflies using CDC light trap from four tourist caves in Northern Thailand. Female sandflies were provisionally sorted into 13 morphospecies and 19 unidentified specimens. DNA was extracted from the thorax and legs of sandflies and the DNA barcode region of cytochrome c oxidase I mtDNA amplified and sequenced. The specimens were sorted into 22 molecular operational taxonomic units (MOTU) based on the 145 DNA barcodes, which is significantly more than the morphospecies. Several of the taxa thought to be present in multiple caves, based on morphospecies sorting, split into cave-specific MOTU which likely represent cryptic species. Several MOTU reported in an earlier study from Wihan Cave, Thailand, were also found in these caves. This supports the use of DNA barcodes to investigate species diversity of sandflies and their useful role in surveillance of sandflies in Thailand.

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.

Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system.

BACKGROUND: Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays. METHODS: A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency's methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays. RESULTS: Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993). CONCLUSION: The presence of (oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries. Detection using qPCR is feasible for quantifying both pathogenic C. parvum and G. lamblia in large water samples.

Towards monitoring the sandflies (Diptera: Psychodidae) of Thailand: DNA barcoding the sandflies of Wihan Cave, Uttaradit.

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.

DISTRIBUTION OF ENTOMOPATHOGENIC NEMATODES IN LOWER NORTHERN THAILAND.

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.

Survey of Angiostrongylus cantonensis in rats and giant African land snails in Phitsanulok province, Thailand.

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.