Strongyloides stercoralis genotyping in a human population in southwestern Iran.

BACKGROUND: Strongyloidiasis is a neglected tropical disease (NTD) that is caused mainly by Strongyloides stercoralis, with an estimated 600 million people infected worldwide, and in fewer cases by Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi. A number of studies have been conducted on the genetic diversity of S. stercoralis in East and Southeast Asia; however, there is very limited corresponding information from West Asian countries, including Iran. METHODS: For Strongyloides worms collected from patients in southwestern Iran, the hypervariable regions I (HVR-I) and IV (HVR-IV) of the nuclear 18S ribosomal DNA (rDNA) locus (SSU) and a fragment of the subunit 1 mitochondrial cytochrome c oxidase gene (cox-1) were sequenced. For a subset of the worms, whole-genome sequencing data were generated. RESULTS: The cox-1 sequences of 136 worms isolated from 23 patients indicated that all isolates were S. stercoralis. Among the cox-1 sequences, 33 polymorphic sites and 13 haplotypes were found. The phylogenetic analysis demonstrated that some sequences clustered fairly closely with sequences from humans and dogs from other parts of the world, while others formed a separate, Iran-specific group. Among 64 S. stercoralis analyzed, we found three of the previously described SSU HVR-I haplotypes, with haplotype II being the most frequent haplotype. In contrast to Southeast Asia, where S. stercoralis heterozygous for different haplotypes at the HVR-I locus are rare, we found 20 worms to be heterozygous for two different HVR-I haplotypes, 18 of which fell into the Iran-specific cox-1 cluster. SSU-heterozygous worms also showed elevated heterozygosity at the whole-genome level. CONCLUSIONS: We conclude that the S. stercoralis population from the Khuzestan province shares much of the genetic diversity with the population in Southeast Asia, but there is an indication of additional genetic input. There appears to be some population structure with different subpopulations, which however do interbreed at least occasionally.

A safety mechanism enables tissue-specific resistance to protein aggregation during aging in C. elegans.

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity.

The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.

Thousands of Pristionchus pacificus orphan genes were integrated into developmental networks that respond to diverse environmental microbiota.

Adaptation of organisms to environmental change may be facilitated by the creation of new genes. New genes without homologs in other lineages are known as taxonomically-restricted orphan genes and may result from divergence or de novo formation. Previously, we have extensively characterized the evolution and origin of such orphan genes in the nematode model organism Pristionchus pacificus. Here, we employ large-scale transcriptomics to establish potential functional associations and to measure the degree of transcriptional plasticity among orphan genes. Specifically, we analyzed 24 RNA-seq samples from adult P. pacificus worms raised on 24 different monoxenic bacterial cultures. Based on coexpression analysis, we identified 28 large modules that harbor 3,727 diplogastrid-specific orphan genes and that respond dynamically to different bacteria. These coexpression modules have distinct regulatory architecture and also exhibit differential expression patterns across development suggesting a link between bacterial response networks and development. Phylostratigraphy revealed a considerably high number of family- and even species-specific orphan genes in certain coexpression modules. This suggests that new genes are not attached randomly to existing cellular networks and that integration can happen very fast. Integrative analysis of protein domains, gene expression and ortholog data facilitated the assignments of biological labels for 22 coexpression modules with one of the largest, fast-evolving module being associated with spermatogenesis. In summary, this work presents the first functional annotation for thousands of P. pacificus orphan genes and reveals insights into their integration into environmentally responsive gene networks.

Chromosome fusions repatterned recombination rate and facilitated reproductive isolation during Pristionchus nematode speciation.

Large-scale genome-structural evolution is common in various organisms. Recent developments in speciation genomics revealed the importance of inversions, whereas the role of other genome-structural rearrangements, including chromosome fusions, have not been well characterized. We study genomic divergence and reproductive isolation of closely related nematodes: the androdioecious (hermaphroditic) model Pristionchus pacificus and its dioecious sister species Pristionchus exspectatus. A chromosome-level genome assembly of P. exspectatus using single-molecule and Hi-C sequencing revealed a chromosome-wide rearrangement relative to P. pacificus. Strikingly, genomic characterization and cytogenetic studies including outgroup species Pristionchus occultus indicated two independent fusions involving the same chromosome, ChrIR, between these related species. Genetic linkage analysis indicated that these fusions altered the chromosome-wide pattern of recombination, resulting in large low-recombination regions that probably facilitated the coevolution between some of the ~14.8% of genes across the entire genomes. Quantitative trait locus analyses for hybrid sterility in all three sexes revealed that major quantitative trait loci mapped to the fused chromosome ChrIR. While abnormal chromosome segregations of the fused chromosome partially explain hybrid female sterility, hybrid-specific recombination that breaks linkage of genes in the low-recombination region was associated with hybrid male sterility. Thus, recent chromosome fusions repatterned recombination rate and drove reproductive isolation during Pristionchus speciation.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2, and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25, and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus.

A New Hope: A Hermaphroditic Nematode Enables Analysis of a Recent Whole Genome Duplication Event.

Whole genome duplication (WGD) is often considered a major driver of evolution that leads to phenotypic novelties. However, the importance of WGD for evolution is still controversial because most documented WGD events occurred anciently and few experimental systems amenable to genetic analysis are available. Here, we report a recent WGD event in the hermaphroditic nematode Allodiplogaster sudhausi and present a comparison with a gonochoristic (male/female) sister species that did not undergo WGD. Self-fertilizing reproduction of A. sudhausi makes it amenable to functional analysis and an ideal system to study WGD events. We document WGD in A. sudhausi through karyotype analysis and whole genome sequencing, the latter of which allowed us to 1) identify functional bias in retention of protein domains and metabolic pathways, 2) show most duplicate genes are under evolutionary constraint, 3) show a link between sequence and expression divergence, and 4) characterize differentially expressed duplicates. We additionally show WGD is associated with increased body size and an abundance of repeat elements (36% of the genome), including a recent expansion of the DNA-hAT/Ac transposon family. Finally, we demonstrate the use of CRISPR/Cas9 to generate mutant knockouts, whereby two WGD-derived duplicate genes display functional redundancy in that they both need to be knocked out to generate a phenotype. Together, we present a novel experimental system that is convenient for examining and characterizing WGD-derived genes both computationally and functionally.

The improved genome of the nematode Parapristionchus giblindavisi provides insights into lineage-specific gene family evolution.

Nematodes such as Caenorhabditis elegans and Pristionchus pacificus are extremely successful model organisms for comparative biology. Several studies have shown that phenotypic novelty but also conserved processes are controlled by taxon-restricted genes. To trace back the evolution of such new or rapidly evolving genes, a robust phylogenomic framework is indispensable. Here, we present an improved version of the genome of Parapristionchus giblindavisi which is the only known member of the sister group of Pristionchus. Relative to the previous short-read assembly, the new genome is based on long reads and displays higher levels of contiguity, completeness, and correctness. Specifically, the number of contigs dropped from over 7,303 to 735 resulting in an N50 increase from 112 to 791 kb. We made use of the new genome to revisit the evolution of multiple gene families. This revealed Pristionchus-specific expansions of several environmentally responsive gene families and a Pristionchus-specific loss of the de novo purine biosynthesis pathway. Focusing on the evolution of sulfatases and sulfotransferases, which control the mouth form plasticity in P. pacificus, reveals differences in copy number and genomic configurations between the genera Pristionchus and Parapristionchus. Altogether, this demonstrates the utility of the P. giblindavisi genome to date and polarizes lineage-specific patterns.

Analysis of repeat elements in the Pristionchus pacificus genome reveals an ancient invasion by horizontally transferred transposons.

BACKGROUND: Repetitive sequences and mobile elements make up considerable fractions of individual genomes. While transposition events can be detrimental for organismal fitness, repetitive sequences form an enormous reservoir for molecular innovation. In this study, we aim to add repetitive elements to the annotation of the Pristionchus pacificus genome and assess their impact on novel gene formation. RESULTS: Different computational approaches define up to 24% of the P. pacificus genome as repetitive sequences. While retroelements are more frequently found at the chromosome arms, DNA transposons are distributed more evenly. We found multiple DNA transposons, as well as LTR and LINE elements with abundant evidence of expression as single-exon transcripts. When testing whether transposons disproportionately contribute towards new gene formation, we found that roughly 10-20% of genes across all age classes overlap transposable elements with the strongest trend being an enrichment of low complexity regions among the oldest genes. Finally, we characterized a horizontal gene transfer of Zisupton elements into diplogastrid nematodes. These DNA transposons invaded nematodes from eukaryotic donor species and experienced a recent burst of activity in the P. pacificus lineage. CONCLUSIONS: The comprehensive annotation of repetitive elements in the P. pacificus genome builds a resource for future functional genomic analyses as well as for more detailed investigations of molecular innovations.

Adaptation to environmental temperature in divergent clades of the nematode Pristionchus pacificus.

Because of ongoing climate change, populations of organisms are being subjected to stressful temperatures more often. This is especially problematic for ectothermic organisms, which are likely to be more sensitive to changes in temperature. Therefore, we need to know if ectotherms have adapted to environmental temperature and, if so, what are the evolutionary mechanisms behind such adaptation. Here, we use the nematode Pristionchus pacificus as a case study to investigate thermal adaptation on the Indian Ocean island of La Réunion, which experiences a range of temperatures from coast to summit. We study the evolution of high-temperature tolerance by constructing a phylogenetic tree of strains collected from many different thermal niches. We show that populations of P. pacificus at low altitudes have higher fertility at warmer temperatures. Most likely, this phenotype has arisen recently and at least twice independently, consistent with parallel evolution. We also studied low-temperature tolerance and showed that populations from high altitudes have increased their fertility at cooler temperatures. Together, these data indicate that P. pacificus strains on La Réunion are subject to divergent selection, adapting to hot and cold niches at the coast and summit of the volcano. Precisely defining these thermal niches provides essential information for models that predict the impact of future climate change on these populations.

Multiple Pristionchus pacificus genomes reveal distinct evolutionary dynamics between de novo candidates and duplicated genes.

The birth of new genes is a major molecular innovation driving phenotypic diversity across all domains of life. Although repurposing of existing protein-coding material by duplication is considered the main process of new gene formation, recent studies have discovered thousands of transcriptionally active sequences as a rich source of new genes. However, differential loss rates have to be assumed to reconcile the high birth rates of these incipient de novo genes with the dominance of ancient gene families in individual genomes. Here, we test this rapid turnover hypothesis in the context of the nematode model organism Pristionchus pacificus We extended the existing species-level phylogenomic framework by sequencing the genomes of six divergent P. pacificus strains. We used these data to study the evolutionary dynamics of different age classes and categories of origin at a population level. Contrasting de novo candidates with new families that arose by duplication and divergence from known genes, we find that de novo candidates are typically shorter, show less expression, and are overrepresented on the sex chromosome. Although the contribution of de novo candidates increases toward young age classes, multiple comparisons within the same age class showed significantly higher attrition in de novo candidates than in known genes. Similarly, young genes remain under weak evolutionary constraints with de novo candidates representing the fastest evolving subcategory. Altogether, this study provides empirical evidence for the rapid turnover hypothesis and highlights the importance of the evolutionary timescale when quantifying the contribution of different mechanisms toward new gene formation.

Sex or cannibalism: Polyphenism and kin recognition control social action strategies in nematodes.

Resource polyphenisms, where single genotypes produce alternative feeding strategies in response to changing environments, are thought to be facilitators of evolutionary novelty. However, understanding the interplay between environment, morphology, and behavior and its significance is complex. We explore a radiation of Pristionchus nematodes with discrete polyphenic mouth forms and associated microbivorous versus cannibalistic traits. Notably, comparing 29 Pristionchus species reveals that reproductive mode strongly correlates with mouth-form plasticity. Male-female species exhibit the microbivorous morph and avoid parent-offspring conflict as indicated by genetic hybrids. In contrast, hermaphroditic species display cannibalistic morphs encouraging competition. Testing predation between 36 co-occurring strains of the hermaphrodite P. pacificus showed that killing inversely correlates with genomic relatedness. These empirical data together with theory reveal that polyphenism (plasticity), kin recognition, and relatedness are three major factors that shape cannibalistic behaviors. Thus, developmental plasticity influences cooperative versus competitive social action strategies in diverse animals.

Single worm transcriptomics identifies a developmental core network of oscillating genes with deep conservation across nematodes.

High-resolution spatial and temporal maps of gene expression have facilitated a comprehensive understanding of animal development and evolution. In nematodes, the small body size represented a major challenge for such studies, but recent advancements have helped overcome this limitation. Here, we have implemented single worm transcriptomics (SWT) in the nematode model organism Pristionchus pacificus to provide a high-resolution map of the developmental transcriptome. We selected 38 time points from hatching of the J2 larvae to young adults to perform transcriptome analysis over 60 h of postembryonic development. A mean sequencing depth of 4.5 million read pairs allowed the detection of more than 23,135 (80%) of all genes. Nearly 3000 (10%) genes showed oscillatory expression with discrete expression levels, phases, and amplitudes. Gene age analysis revealed an overrepresentation of ancient gene classes among oscillating genes, and around one-third of them have 1:1 orthologs in C. elegans One important gene family overrepresented among oscillating genes is collagens. Several of these collagen genes are regulated by the developmental switch gene eud-1, indicating a potential function in the regulation of mouth-form plasticity, a key developmental process in this facultative predatory nematode. Together, our analysis provides (1) an updated protocol for SWT in nematodes that is applicable to many microscopic species, (2) a 1- to 2-h high-resolution catalog of P. pacificus gene expression throughout postembryonic development, and (3) a comparative analysis of oscillatory gene expression between the two model organisms P. pacificus and C. elegans and associated evolutionary dynamics.

Nine new Pristionchus (Nematoda: Diplogastridae) species from China.

The model organism Pristionchus pacificus and the genus Pristionchus, Kreis, 1932 have been intensively studied in the last decade with contemporary work focusing on the development, evolution, ecology, behavior, neurobiology, and genomics of this group of organisms. In particular, mechanistic studies on the development and evolution of mouth-form plasticity, predation and associated self-recognition processes enabled unique insight into life history strategies and the evolution of novelty. These studies include a comparative research agenda making use of the 39 available species of Pristionchus, all of which can be studied in living cultures. Sampling efforts revealed that Asia represents a biodiversity hotspot for Pristionchus worms. However, previous samplings have a bias towards northern and island areas, largely for logistic reasons. Here, we report on two extensive sampling trips to the Yunnan and Shaanxi provinces in Mainland China. We report the isolation of nine new Pristionchus species by morphology, morphometrics, mating experiments and genome-wide sequence analysis.

The community-curated Pristionchus pacificus genome facilitates automated gene annotation improvement in related nematodes.

BACKGROUND: The nematode Pristionchus pacificus is an established model organism for comparative studies with Caenorhabditis elegans. Over the past years, it developed into an independent animal model organism for elucidating the genetic basis of phenotypic plasticity. Community-based curations were employed recently to improve the quality of gene annotations of P. pacificus and to more easily facilitate reverse genetic studies using candidate genes from C. elegans. RESULTS: Here, I demonstrate that the reannotation of phylogenomic data from nine related nematode species using the community-curated P. pacificus gene set as homology data substantially improves the quality of gene annotations. Benchmarking of universal single copy orthologs (BUSCO) estimates a median completeness of 84% which corresponds to a 9% increase over previous annotations. Nevertheless, the ability to infer gene models based on homology already drops beyond the genus level reflecting the rapid evolution of nematode lineages. This also indicates that the highly curated C. elegans genome is not optimally suited for annotating non-Caenorhabditis genomes based on homology. Furthermore, comparative genomic analysis of apparently missing BUSCO genes indicates a failure of ortholog detection by the BUSCO pipeline due to the insufficient sample size and phylogenetic breadth of the underlying OrthoDB data set. As a consequence, the quality of multiple divergent nematode genomes might be underestimated. CONCLUSIONS: This study highlights the need for optimizing gene annotation protocols and it demonstrates the benefit of a high quality genome for phylogenomic data of related species.

Spatial Transcriptomics of Nematodes Identifies Sperm Cells as a Source of Genomic Novelty and Rapid Evolution.

Divergence of gene function and expression during development can give rise to phenotypic differences at the level of cells, tissues, organs, and ultimately whole organisms. To gain insights into the evolution of gene expression and novel genes at spatial resolution, we compared the spatially resolved transcriptomes of two distantly related nematodes, Caenorhabditis elegans and Pristionchus pacificus, that diverged 60-90 Ma. The spatial transcriptomes of adult worms show little evidence for strong conservation at the level of single genes. Instead, regional expression is largely driven by recent duplication and emergence of novel genes. Estimation of gene ages across anatomical structures revealed an enrichment of novel genes in sperm-related regions. This provides first evidence in nematodes for the "out of testis" hypothesis that has been previously postulated based on studies in Drosophila and mammals. "Out of testis" genes represent a mix of products of pervasive transcription as well as fast evolving members of ancient gene families. Strikingly, numerous novel genes have known functions during meiosis in Caenorhabditis elegans indicating that even universal processes such as meiosis may be targets of rapid evolution. Our study highlights the importance of novel genes in generating phenotypic diversity and explicitly characterizes gene origination in sperm-related regions. Furthermore, it proposes new functions for previously uncharacterized genes and establishes the spatial transcriptome of Pristionchus pacificus as a catalog for future studies on the evolution of gene expression and function.

Rhabditophanes diutinus a parthenogenetic clade IV nematode with dauer larvae.

Comparative studies using non-parasitic model species such as Caenorhabditis elegans, have been very helpful in investigating the basic biology and evolution of parasitic nematodes. However, as phylogenetic distance increases, these comparisons become more difficult, particularly when outside of the nematode clade to which C. elegans belongs (V). One of the reasons C. elegans has nevertheless been used for these comparisons, is that closely related well characterized free-living species that can serve as models for parasites of interest are frequently not available. The Clade IV parasitic nematodes Strongyloides are of great research interest due to their life cycle and other unique biological features, as well as their medical and veterinary importance. Rhabditophanes, a closely related free-living genus, forms part of the Strongyloidoidea nematode superfamily. Rhabditophanes diutinus (= R. sp. KR3021) was included in the recent comparative genomic analysis of the Strongyloididae, providing some insight into the genomic nature of parasitism. However, very little is known about this species, limiting its usefulness as a research model. Here we provide a species description, name the species as R. diutinus and investigate its life cycle and subsequently gene expression in multiple life stages. We identified two previously unreported starvation induced life stages: dauer larvae and arrested J2 (J2A) larvae. The dauer larvae are morphologically similar to and are the same developmental stage as dauers in C. elegans and infective larvae in Strongyloides. As in C. elegans and Strongyloides, dauer formation is inhibited by treatment with dafachronic acid, indicating some genetic control mechanisms are conserved. Similarly, the expression patterns of putative dauer/infective larva control genes resemble each other, in particular between R. diutinus and Strongyloides spp. These findings illustrate and increase the usefulness of R. diutinus as a non-parasitic, easy to work with model species for the Strongyloididae for studying the evolution of parasitism as well as many aspects of the biology of Strongyloides spp, in particular the formation of infective larvae.

Comparative genomics and community curation further improve gene annotations in the nematode Pristionchus pacificus.

BACKGROUND: Nematode model organisms such as Caenorhabditis elegans and Pristionchus pacificus are powerful systems for studying the evolution of gene function at a mechanistic level. However, the identification of P. pacificus orthologs of candidate genes known from C. elegans is complicated by the discrepancy in the quality of gene annotations, a common problem in nematode and invertebrate genomics. RESULTS: Here, we combine comparative genomic screens for suspicious gene models with community-based curation to further improve the quality of gene annotations in P. pacificus. We extend previous curations of one-to-one orthologs to larger gene families and also orphan genes. Cross-species comparisons of protein lengths, screens for atypical domain combinations and species-specific orphan genes resulted in 4311 candidate genes that were subject to community-based curation. Corrections for 2946 gene models were implemented in a new version of the P. pacificus gene annotations. The new set of gene annotations contains 28,896 genes and has a single copy ortholog completeness level of 97.6%. CONCLUSIONS: Our work demonstrates the effectiveness of comparative genomic screens to identify suspicious gene models and the scalability of community-based approaches to improve the quality of thousands of gene models. Similar community-based approaches can help to improve the quality of gene annotations in other invertebrate species, including parasitic nematodes.

Extracellular proteostasis prevents aggregation during pathogenic attack.

In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1-5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6-9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.

Genomic studies on Strongyloides stercoralis in northern and western Thailand.

BACKGROUND: Strongyloidiasis is a soil borne helminthiasis, which in most cases is caused by Strongyloides stercoralis. Human infections with S. fuelleborni fuelleborni and S. fuelleborni kellyi also occur. Although up to 370 million people are currently estimated to be infected with S. stercoralis, this parasite is frequently overlooked. Strongyloides stercoralis is prevalent among humans in Thailand; however, S. fuelleborni fuelleborni has also been reported. Three recent genomic studies of individual S. stercoralis worms found genetically diverse populations of S. stercoralis, with comparably low heterozygosity in Cambodia and Myanmar, and less diverse populations with high heterozygosity in Japan and southern China that presumably reproduce asexually. METHODS: We isolated individual Strongyloides spp. from different localities in northern and western Thailand and determined their nuclear small ribosomal subunit rDNA (18S rDNA, SSU), in particular the hypervariable regions I and IV (HVR-I and HVR-IV), mitochondrial cytochrome c oxidase subunit 1 (cox1) and for a subset whole genome sequences. These sequences were then compared with each other and with published sequences from different geographical locations. RESULTS: All 237 worms isolated from 16 different human hosts were S. stercoralis, no S. fuelleborni was found. All worms had the common S. stercoralis SSU HVR IV haplotype A. Two different SSU HVR I haplotypes (I and II), both previously described in S. stercoralis, were found. No animal heterozygous for the two haplotypes was identified. Among the twelve cox1 haplotypes found, five had not been previously described. Based upon the mitochondrial cox1 and the nuclear whole genome sequences, S. stercoralis in Thailand was phylogenetically intermixed with the samples from other Southeast Asian countries and did not form its own branch. The genomic heterozygosity was even slightly lower than in the samples from the neighboring countries. CONCLUSIONS: In our sample from humans, all Strongyloides spp. were S. stercoralis. The S. stercoralis from northern and western Thailand appear to be part of a diverse, intermixing continental Southeast Asian population. No obvious indication for genetic sub-structuring of S. stercoralis within Thailand or within the Southeast Asian peninsula was detected.