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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.

Summary Entomopathogenic nematodes (EPN) Steinernema and Heterorhabditis with symbionts with Xenorhabdus and Photorhabdus bacteria, respectively, are reported as biocontrol agents for insect control. The objectives of this study were to identify EPN and their symbiotic bacteria in national parks of Phitsanulok Province, Thailand, and to test how Xenorhabdus isolates attack Aedes aegypti larvae. We collected 810 soil samples from four national parks. The juvenile stage of EPN was isolated from soil samples using a baiting technique with Galleria mellonella followed by a White trap. Partial regions of 28S rDNA and internal transcript spacer were sequenced to identify EPN, and recA sequencing was used to discriminate between Xenorhabdus and Photorhabdus. We found that 74 of the 810 soil samples (9.1%) were positive for the EPN. The EPN were molecularly identified as S. surkhetense, S. longicaudum, H. indica and Heterorhabditis sp. SGmg3, and their symbiotic bacteria were identified as X. stockiae, X. griffiniae, X. indica, X. vietnamensis, P. luminescens subsp. akhurstii, and P. temperata subsp. temperata. Xenorhabdus griffiniae showed potential larvicidal activity against A. aegypti (91% mortality at 72 and 96 h after exposure). This study demonstrates the diversity of EPN and symbiotic bacteria in national parks of Thailand and the potential to use X. griffiniae as a biocontrol agent to kill A. aegypti larvae.

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.

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 (H

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.

Entomopathogenic nematodes (EPNs) Steinernema and Heterorhabditis and their symbiotic bacteria, Xenorhabdus and Photorhabdus, have been successfully used for the control of insect pests. The objectives of this study were to survey the EPNs and symbiotic bacteria in the agricultural areas of the Phitsanulok province, Thailand, and to study the association between the soil parameters and presence of EPNs. We collected 200 soil samples from 40 soil sites in agricultural areas (field crops, horticulture crops and forest). The prevalence of EPNs was 8.0% (16/200). Fifteen of the EPN isolates were molecularly identified (based on 28S ribosomal DNA and internal transcribed spacer regions) as Steinernema siamkayai. Seven isolates of Xenorhabdus stockiae were identified using recombinase A sequencing. Phylogenetic analysis revealed that all the Steinernema and Xenorhabdus isolates were closely related to S. siamkayai (Indian strain) and X. stockiae (Thai strain), respectively. Significantly more EPNs were recovered from loam than from clay. Although the association between soil parameters (pH, temperature and moisture) and the presence of EPNs was not statistically significant, the elevation levels of the soil sites with and without EPNs were found to be different. Moreover, statistical comparisons between the agricultural areas revealed no significant differences. Therefore, we concluded that S. siamkayai is associated with X. stockiae in agricultural areas and that there is no association between the soil parameters of agricultural areas and presence of EPNs, except for soil texture and the elevation. Steinernema siamkayai may be applied as a biocontrol agent in agricultural areas.

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.

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.

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.