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.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

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.

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.

Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand.

Bacteria of the genera Photorhabdus and Xenorhabdus produce a plethora of natural products to support their similar symbiotic life cycles. For many of these compounds, the specific bioactivities are unknown. One common challenge in natural product research when trying to prioritize research efforts is the rediscovery of identical (or highly similar) compounds from different strains. Linking genome sequence to metabolite production can help in overcoming this problem. However, sequences are typically not available for entire collections of organisms. Here, we perform a comprehensive metabolic screening using HPLC-MS data associated with a 114-strain collection (58 Photorhabdus and 56 Xenorhabdus) across Thailand and explore the metabolic variation among the strains, matched with several abiotic factors. We utilize machine learning in order to rank the importance of individual metabolites in determining all given metadata. With this approach, we were able to prioritize metabolites in the context of natural product investigations, leading to the identification of previously unknown compounds. The top three highest ranking features were associated with Xenorhabdus and attributed to the same chemical entity, cyclo(tetrahydroxybutyrate). This work also addresses the need for prioritization in high-throughput metabolomic studies and demonstrates the viability of such an approach in future research.

Photorhabdusluminescens subsp. namnaonensis subsp. nov., isolated from Heterorhabditisbaujardi nematodes.

A lightly yellowish-pigmented, oxidase-negative bacterial strain (PB45.5T) isolated from the Nam Nao district of Phetchabun in central Thailand was investigated to determine its taxonomic position. Cells of the isolate showed a rod shaped appearance. The strain stained Gram-negative. Strain PB45.5T shared highest 16S rRNA gene sequence similarity with the type strains of Photorhabdus luminescens subsp. akhurstii (99.2 %) and Photorhabdus luminescens subsp. hainanensis (99.1 %) and lower similarities to all other Photorhabdus luminescens subspecies (<98.0 %). Multilocus sequence analysis (MLSA) based on concatenated partial recA, dnaN, gltX, gyrB and infB gene sequences confirmed the affiliation obtained by 16S rRNA gene sequence analysis but showed a clear distinction of PB45.5T from the closest related type strains. Strain PB45.5T shared only 96.9 % sequence similarity (concatenated nucleotide sequences) with P. luminescens subsp. akhurstii FRG04T and 96.8 % with P. luminescens subsp. hainanensis C8404T. The fatty acid profile of the strain consisted of the major fatty acids C14 : 0, C16 : 0, C17 : 0 cyclo, C16 : 1ω7c and/or iso-C15 : 0 2-OH, and C18 : 1ω7c. The MLSA results and the differential biochemical and chemotaxonomic properties showed that strain PB45.5T represents a novel P. luminescens subspecies, for which the name Photorhabdus luminescens subsp. namnaonensis subsp. nov. (type strain PB45.5T=LMG 29915T=CCM 8729T) is proposed.

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.

A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase.

Simple urea compounds ("phurealipids") have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

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.

Biosynthesis of the insecticidal xenocyloins in Xenorhabdus bovienii.

The biosynthesis gene cluster for the production of xenocyloins was identified in the entomopathogenic bacterium Xenorhabdus bovienii SS-2004, and their biosynthesis was elucidated by heterologous expression and in vitro characterization of the enzymes. XclA is an S-selective ThDP-dependent acyloin-like condensation enzyme, and XclB and XclC are examples of the still-rare acylating ketosynthases that catalyze the acylation of the XclA-derived initial xenocyloins with acetyl-, propionyl-, or malonyl-CoA, thereby resulting in the formation of further xenocyloin derivatives. All xenocyloins were produced mainly by the more virulent primary variant of X. bovienii and showed activity against insect hemocytes thus contributing to the overall virulence of X. bovienii against insects.

Antiparasitic chaiyaphumines from entomopathogenic Xenorhabdus sp. PB61.4.

A new class of four depsipentapeptides called chaiyaphumines A-D (1-4) was isolated from Xenorhabdus sp. PB61.4. Their structures were elucidated by detailed 1D and 2D NMR experiments and by a Marfey's analysis following flash hydrolysis of the peptide. Verification of the structure was achieved by three-dimensional modeling using NOE-derived distance constraints, molecular dynamics, and energy minimization. Chaiyaphumine A (1) showed good activity against Plasmodium falciparum (IC50 of 0.61 µM), the causative agent of malaria, and was active against other protozoal tropical disease causing agents.

Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei.

Known mechanisms of resistance to ß-lactam antibiotics include ß-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of ß-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water.

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.

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.

Genetic diversity and population structure of Physella acuta (Gastropoda: Physidae) in Thailand using mitochondrial gene markers: COI and 16S rDNA.

Physella acuta is a freshwater snail native to North America. Understanding the phylogeography and genetic structure of P. acuta will help elucidate its evolution. In this study, we used mitochondrial (COI and 16S rDNA) and nuclear (ITS1) markers to identify the species and examine its genetic diversity, population structure, and demographic history of P. acuta in Thailand. Phylogenetic and network analyses of P. acuta in Thailand pertained to clade A, which exhibits a global distribution. Analysis of the genetic structure of the population revealed that the majority of pairwise comparisons showed no genetic dissimilarity. An isolation-by-distance test indicates no significant correlation between genetic and geographical distances among P. acuta populations, suggesting that gene flow is not restricted by distance. Demographic history and haplotype network analyses suggest a population expansion of P. acuta, as evidenced by the star-like structure detected in the median-joining network. Based on these results, we concluded that P. acuta in Thailand showed gene flow and recent population expansion. Our findings provide fundamental insights into the genetic variation of P. acuta in Thailand.

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.

Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

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.

Genome mining reveals novel biosynthetic gene clusters in entomopathogenic bacteria.

The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.