STEINERNEMA ADAMSI N. SP. (RHABDITIDA: STEINERNEMATIDAE), A NEW ENTOMOPATHOGENIC NEMATODE FROM THAILAND.

A new species of entomopathogenic nematode, Steinernema adamsi n. sp., was recovered from the soil of a longan tree (Dimocarpus sp.) in Mueang Lamphun District, Thailand, using baiting techniques. Upon analysis of the nematode's morphological traits, we found it to be a new species of Steinernema and a member of the Longicaudatum clade. Molecular analyses of the ITS rDNA and D2D3 of 28S rDNA sequences further confirmed that S. adamsi n. sp. is a new species of the Longicaudatum clade, which is closely related to Steinernema guangdongense and Steinernema longicaudam. Using morphometric analysis, the infective juveniles measure between 774.69 and 956.96 µm, males have a size range of 905.44 to 1,281.98 µm, and females are within the range of 1,628.21 to 2,803.64 µm. We also identified the symbiotic bacteria associated with the nematode based on 16S sequences as Xenorhabdus spp. closely related toXenorhabdus griffiniae. Furthermore, we have successfully assessed a cryopreservation method for the long-term preservation of S. adamsi n. sp. Successful cryopreservation of this new species will allow for the longer preservation of its traits and will be valuable for its future use. The discovery of this new species has significant implications for the development of effective biological control agents in Thailand, and our work contributes to our understanding of the diversity and evolution of entomopathogenic nematodes.

Assessment of the genetic diversity of lymnaeid (Gastropoda: Pulmonata) snails and their infection status with trematode cercariae in different regions of Thailand.

Lymnaeid snails are some of the most widespread snails and are the first intermediate host of trematode parasites that affect human and livestock health. A full understanding of the genetic relationship of hosts and parasites is of paramount importance for effective parasite management. The present study assessed the prevalence of trematode larvae in lymnaeid snails and examined the genetic diversity of these snails collected across Thailand. We collected 672 lymnaeid snails from 39 locations in 22 provinces of six regions in Thailand. Subsequently, cercarial infection in the snails was observed by using the shedding method. Lymnaeid snails released 5 types of trematode cercariae, namely, xiphidiocercariae, echinostome cercariae I, echinostome cercariae II, furcocercous cercariae, and strigea cercariae. The phylogenetic analysis based on ITS2 and 28S rDNA sequences revealed 5 cercaria types assigned to four trematode families, of which two belong to the group of human intestinal flukes. Combination of shell morphology and sequence analysis of the mitochondrial COI and 16S rDNA genes, the lymnaeid snails were classified into two species, Radix rubiginosa and Orientogalba viridis. Moreover, the combined dataset of mtDNA genes (COI + 16S rDNA) from R. rubiginosa and O. viridis revealed 32 and 15 different haplotypes, respectively, of which only a few haplotypes were infected with cercariae. The genetic diversity and genetic structure revealed that R. rubiginosa and O. viridis experienced a bottleneck phenomenon, and showed limited gene flow between populations. Population demographic history analyses revealed that R. rubiginosa and O. viridis experienced population reductions followed by recent population expansion. These findings may improve our understanding of parasite-lymnaeid evolutionary relationships, as well as the underlying molecular genetic basis, which is information that can be used for further effective control of the spread of trematode disease.

Genetic variation of the freshwater snail Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand, inferred from 18S and 28S rDNA sequences.

Indoplanorbis exustus, a freshwater pulmonate snail, is widely distributed in tropical and subtropical zones and plays a significant role as an intermediate host for trematode parasites. Various genetic markers have been used for species identification and phylogenetic studies of this snail. However, there are limited studies about their molecular genetics based on nuclear ribosomal DNA (rDNA) genes. A genetic analysis of I. exustus in Thailand was conducted based on the nuclear 18S rDNA (339 bp) and 28S rDNA (1036 bp) genes. Indoplanorbis snails were collected from 29 localities in 21 provinces covering six regions of Thailand. Nucleotide sequences from 44 snails together with sequences from the GenBank database were examined for phylogenetic relationships and genetic diversity. All sequences of the selected nucleotide regions exhibited a high level of similarity (99%) to the sequences of I. exustus in the GenBank database. The maximum likelihood tree based on the 18S and 28S rDNA fragment sequences of I. exustus in Thailand revealed only one group with clear separation from another genus in the family Planorbidae. The I. exustus 28S rDNA sequences showed intraspecific genetic divergence ranging from 0 to 0.78% and were classified into 8 different haplotypes. Conversely, the 18S rDNA data showed lower variation than the 28S rDNA data and revealed a single haplotype and intraspecific distances of zero among all sampled individuals. The haplotype network of 28S rDNA sequences of I. exustus in Thailand revealed six unique haplotypes and two haplotypes shared by at least two regions. Overall, both markers were successful in the identification of I. exustus. However, these markers, particularly the 18S rDNA, may not be suitable for genetic analysis within the species, particularly for population genetic studies, due to their limited variation as seen in this study. In summary, this study not only enhances understanding of genetic variation in I. exustus but is also useful for the selection of molecular markers in future genetic research.

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.

Entomopathogenic nematodes isolated from agricultural areas of Thailand and their activity against the larvae of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have been considered to be effective biological control agents for several insects. In this study, we isolated and identified EPNs from soil samples in agricultural areas of northern Thailand and evaluated their efficacy for controlling larvae of three mosquito vector species, Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. A total of 51 of 1,000 soil samples were positive (5.1% prevalence) for EPNs, which were identified through sequencing of the rDNA and ITS to 37 Steinernema isolates (3.7%) and 14 Heterorhabditis isolates (1.4%). For the bioassay, the larvae of mosquitoes were exposed to Steinernema surkhetense (eALN6.3_TH), Steinernema lamjungense (eALN11.5_TH), Heterorhabditis indica (eACM14.2_TH) and Heterorhabditis bacteriophora (eALN18.2_TH). Heterorhabditis bacteriophora showed the highest efficacy against Ae. aegypti and Cx. quinquefasciatus. At 96 h after exposure, the mortality rates were 60.0 and 91.7%, respectively. The EPNs were observed in the dead mosquito larvae, which were mostly found in the thorax followed by the head and abdomen. Some EPNs were dead with melanization, and some were able to survive in the cavity of mosquito larvae. Our results show the low prevalence of EPN in agricultural areas of Thailand. Moreover, H. bacteriophora may be considered an alternative biocontrol agent for managing and controlling these vector mosquitoes.

Genetic analysis of a 66-kDa protein-encoding gene of Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

The rat lungworm Angiostrongylus cantonensis is globally known to be the cause of oeosinophilic meningitis in humans. Another congener, Angiostrongylus malaysiensis, is closely related to A. cantonensis and has been described as a potential human pathogenic parasite. These 2 worms are similar in terms of life cycle, host range and morphological and genetic information. However, there are limited studies about their genetic diversity based on the 66-kDa protein-encoding gene. The objective of this study was to explore the 66-kDa protein sequence variation of A. cantonensis and A. malaysiensis collected from Thailand. Two adult and 53 third-stage larval specimens of Angiostrongylus from 4 geographic locations in Thailand were molecularly identified using the 66-kDa protein gene. The phylogenetic trees (Bayesian inference tree and maximum-likelihood tree) showed that Angiostrongylus formed a monophyletic clade with a clear separation between A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis varies from 0.82 to 2.86%, with a total of 16 variable sites. The analysis of genetic diversity revealed 1 and 5 new haplotypes of A. cantonensis and A. malaysiensis, respectively, and showed genetic differences between the populations of A. cantonensis and A. malaysiensis. The haplotype networks of A. cantonensis and A. malaysiensis populations in Thailand are similar to those of populations in some countries, indicating the range expansion of genomic origin between populations in different areas. In conclusion, the 66-kDa protein gene was a good genetic marker for studying genetic diversity and discriminating between A. cantonensis and A. malaysiensis.

Entomopathogenic Nematodes and Their Symbiotic Bacteria from the National Parks of Thailand and Larvicidal Property of Symbiotic Bacteria against Aedes aegypti and Culex quinquefasciatus.

Entomopathogenic nematodes (EPNs) are insect parasitic nematodes of the genera Het-erorhabditis and Steinernema. These nematodes are symbiotically associated with the bacteria, Photorhabdus and Xenorhabdus, respectively. National parks in Thailand are a potentially rich resource for recovering native EPNs and their symbiotic bacteria. The objectives of this study are to isolate and identify EPNs and their bacterial flora from soil samples in four national parks in Thailand and to evaluate their efficacy for controlling mosquito larvae. Using a baiting method with a Galleria mellonella moth larvae and a White trap technique, 80 out of 840 soil samples (9.5%) from 168 field sites were positive for EPNs. Sequencing of an internal transcribed spacer resulted in the molecular identification of Heterorhabditis nematode isolates as H. indica, H. baujardi and Heterorhabditis SGmg3, while using 28S rDNA sequencing, Steinernema nematode species were identified as S. guang-dongense, S. surkhetense, S. minutum, S. longicaudum and one closely related to S. yirgalemense. For the symbiotic bacterial isolates, based on recA sequencing, the Photorhabdus spp. were identified as P. luminescens subsp. akhurstii, P. luminescens subsp. hainanensis and P. luminescens subsp. australis. Xenorhabdus isolates were identified as X. stockiae, X. indica, X. griffiniae, X. japonica and X. hominickii. Results of bioassays demonstrate that Photorhabdus isolates were effective on both Aedes aegypti and Culex quinquefasciatus. Therefore, we conclude that soil from Thailand's national parks contain a high diversity of entomopathogenic nematodes and their symbiotic bacteria. Photorhabdus bacteria are larvicidal against culicine mosquitoes and may serve as effective biocontrol agents.

Prevalence and genetic analysis of Enterobius vermicularis in schoolchildren in lower northern Thailand.

Enterobius vermicularis, a nematode parasite with a global distribution causes enterobiasis in schoolchildren and is considered a neglected parasite. An understanding of the prevalence and genetic diversity of enterobiasis is crucial for appropriate control measures. Therefore, the objectives of this research were to study the prevalence and genetic diversity of E. vermicularis in schoolchildren from lower northern Thailand, based on cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) sequences. Using the scotch tape technique, 7.4% (188/2544) of schoolchildren from 21 primary schools were found positive for E. vermicularis eggs, which is a relatively low infection rate. Phylogenetic trees of partial COI sequences (397 bp) revealed similar topologies using maximum likelihood (ML) and neighbor-joining (NJ) methods and identified E. vermicularis type A (105 sequences) and B (1 sequence). Haplotype network analysis of the COI sequences demonstrated a high haplotype diversity (Hd = 0.9028). In contrast, phylogenetic analysts of a 343 bp region of the ITS2 locus (52 sequences) revealed a monophyletic group. More sequence analyses of E. vermicularis from humans and other hosts in Thailand are necessary to better understand the genetic diversity of this parasite.

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.

Preliminary Survey of Larval Trematodes in Freshwater Snails of Phitsanulok Province in Lower Northern Thailand.

Background: Freshwater snails serve as intermediate hosts for a variety of trematodes that cause illness in the human and animal populations. Several species of freshwater snails in Thailand have been found to have larval trematode infections. We aimed to investigate a freshwater snail in Phitsanulok Province and report on its current status of larval trematode infection. Methods: Freshwater snails were collected from six localities (rice field and irrigation canal) by handpicking and using a count per unit of time sampling approach. The snails were identified by their external shell morphology. The shedding method was applied to observe the cercariae, which were photographed under a light microscope to determine their morphological types. Results: A total of 211 snails were classified into seven genera. The most abundant snail species was Lymnaea sp., representing 31.3% of the sample, followed by Physella sp., Bithynia sp., Pomacea canaliculata, Filopaludina martensi, Indoplanorbis exustus, and Melanoides tuberculata, in that order. From the sample, 21 snails (9.95%), including Bithynia sp., Lymnaea sp., I. exustus, and M. tuberculata, were infected with cercarial trematodes, which could be categorized into four types, namely amphistome, parapleurolophocercous, echinostome, and xiphidiocercaria. Amphistome emerged from Bithynia sp., and I. exustus was the most common cercaria to be recovered, representing 80.9% of all infected snails. Conclusion: This study presents the current prevalence of cercariae in infected snails within the studied area. It is important to manage intermediate host snails in order to restrict trematode life cycle completion.

Molecular identification and phylogeny of Steinernema and Heterorhabditis nematodes and their efficacy in controlling the larvae of Aedes aegypti, a major vector of the dengue virus.

Aedes aegypti is the mosquito vector of several arboviruses, especially the dengue virus. Aedes aegypti strain resistant to chemical insecticides have been reported worldwide. To tackle this, an entomopathogenic nematode (EPN) may be an alternative bio-control agent. To this end, this study aims to isolate, identify, and analyze the phylogeny of EPNs in Thailand and evaluate their efficacy for controlling the Ae. aegypti larvae. From 12 provinces in Thailand, soil samples were randomly collected, with 118 out of 1,100 them being positive for EPNs (10.73% prevalence) in genera Steinernema (4.46%) and Heterorhabditis (6.27%). Then, molecular discrimination of these two genus was performed based on the sequencing and phylogenetic analysis of the 28S rDNA and internal transcribed spacer regions. The most abundant species of EPN were Heterorhabditis indica, with minor species of Heterorhabditis sp. SGmg3, H. baujardi, S. surkhetense, S. kushidai, S. siamkayai, Steinernema sp. YNd80, Steinernema sp. YNc215, S. guangdongense, and S. huense. The larvicidal activity of five selected EPN isolates were tested against Ae. aegypti. Ten larvae of Ae. aegypti were incubated with different concentration (80, 160, 320, and 640 IJs/larva) of the infective juveniles of EPN in a 24-well and 6-well plates for 4 days. The mortality rates of the larvae were observed daily. Steinernema surkhetense (ePYO8.5_TH) showed the potential to kill mosquito larvae, with the highest mortality rate of 92 ± 9.37% and 89 ± 9.91% after it was treated with 640 IJs/larva in a 24-well plate and 1600 IJs/larva in a 6-well plate, respectively. There is an abundant distribution of EPNs across the country, and S. surkhetense ePYO8.5_TH may be used as a biocontrol agent against Ae. aegypti 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.

Low genetic diversity and the phylogeny of Achatina fulica, an intermediate host of Angiostrongylus cantonensis in Thailand, inferred from 16S mitochondrial sequences.

The giant African land snail, Achatina fulica, is a pulmonate land snail of the Achatinidae family. The snail is native of East Africa and has spread to tropical and subtropical areas in different parts of the world. Achatina fulica is an important agricultural pests and the intermediate host for Angiostrongylus spp., especially Angiostrongylus cantonensis, which causes eosinophilic meningoencephalitis in humans. However, information on the genetic diversity and phylogenetic relationships among A. fulica populations in Thailand is limited. The goal of this study was to evaluate the genetic diversity and analyze haplotype network relationships between A. fulica populations in Thailand and other areas of the world. We collected A. fulica from 22 provinces in 6 regions of Thailand and analyzed a phylogenetic tree and haplotype network based on a fragment of 239 base pairs of the 16S rRNA sequence. The maximum likelihood, neighbor joining, and Bayesian inference trees for the 133 A. fulica 16S rRNA sequences revealed only one group, closely related only to A. fulica haplotype C, in all regions of Thailand. The haplotype network analysis showed that haplotype C is associated with F, P, E, Q, H and D. The genetic distance between the haplotype and A. fulica isolates in Thailand varies from 0 and 0.024, with a total of 21 variable sites. Haplotype C is predominant in Thailand and associated with other haplotypes from several areas. As inferred from 16S rDNA sequences, this study demonstrated low genetic diversity in A. fulica in Thailand. Low genetic diversity in the population can increase susceptibility to A. cantonensis infection, which may be associated with the current distribution of A. cantonensis.

Genetic analysis of Cryptozona siamensis (Stylommatophora, Ariophantidae) populations in Thailand using the mitochondrial 16S rRNA and COI sequences.

Cryptozona siamensis, one of the most widespread land snails, is native to Thailand, and plays a key role as an agricultural pest and intermediate host for Angiostrongylus spp. However, its genetic diversity and population structure has not yet been investigated, and are poorly understood. Therefore, a genetic analysis of the C. siamensis population in Thailand was conducted, based mitochondrial 16S rRNA (402 bp) and COI (602 bp) gene fragment sequences. Cryptozona siamensis randomly collected from 17 locations in four populations across Thailand, between May 2017 and July 2018. Fifty-eight snails were used to examine the phylogeny, genetic diversity, and genetic structure. The maximum likelihood tree based on the 16S rRNA and COI fragment sequences revealed two main clades. A total of 14 haplotypes with 44 nucleotide variable sites were found in the 16S rRNA sequences, while 14 haplotypes with 57 nucleotide variable sites were found in the COI sequences. The genetic diversity of C. siamensis in term of the number of haplotypes and haplotype diversity, was found to be high but the nucleotide diversity showed low levels of genetic differentiation for the COI sequence as also noted with the 16S rRNA sequence. The population genetic structure of C. siamensis revealed genetic difference in most populations in Thailand. However, low genetic difference in some populations may be due to high gene flow. This study provides novel insights into the basic molecular genetics of C. siamensis.

Genetic characterization of Angiostrongylus larvae and their intermediate host, Achatina fulica, in Thailand.

Angiostrongyliasis is a parasitic disease caused by nematodes of the genus Angiostrongylus. Distribution of this worm corresponds to the dispersal of its main intermediate host, the giant African land snail Achatina fulica. Genetic characterization can help identify parasitic pathogens and control the spreading of disease. The present study describes infection of A. fulica by Angiostrongylus, and provides a genetic outlook based on sequencing of specific regions. We collected 343 land snails from 22 provinces across six regions of Thailand between May 2017 and July 2018. Artificial digestion and Baermann's technique were employed to isolate Angiostrongylus larvae. The worm and its intermediate host were identified by sequencing with specific nucleotide regions. Phylogenetic tree was constructed to evaluate the relationship with other isolates. A. fulica from Chaiyaphum province was infected with A. cantonensis, whereas snails collected from Phrae and Chiang Rai provinces were infected with A. malaysiensis. The maximum likelihood tree based on 74 A. fulica COI sequences revealed monophyletic groups and identified two haplotypes: AF1 and AF2. Only AF1, which is distributed in all regions of Thailand, harbored the larvae of A. cantonensis and A. malaysiensis. Two mitochondrial genes (COI and cytb) and two nuclear regions (ITS2 and SSU rRNA) were sequenced in 41 Angiostrongylus specimens. The COI gene indicated that A. cantonensis was closely related to the AC10 haplotype; whereas the cytb gene revealed two new haplotypes: AC19 and AC20. SSU rRNA was useful for the identification of A. cantonensis; whereas ITS2 was a good genetic marker for differentiating between A. cantonensis and A. malaysiensis. This study provides genetic information about the parasite Angiostrongylus and its snail intermediate host. The data in this work may be useful for further study on the identification of Angiostrongylus spp., the genetic relationship between intermediate host and parasite, and control of parasites.