Free-living Generation Research Articles

The generation of stable transgenic lines in the human-infective nematode Strongyloides stercoralis.

The skin-penetrating gastrointestinal parasitic nematode Strongyloides stercoralis causes strongyloidiasis, which is a neglected tropical disease that is associated with severe chronic illness and fatalities. Unlike other human-infective nematodes, S. stercoralis cycles through a single free-living generation and thus serves as a genetically tractable model organism for understanding the mechanisms that enable parasitism. Techniques such as CRISPR/Cas9-mediated mutagenesis and transgenesis are now routinely performed in S. stercoralis by introducing exogenous DNA into free-living adults and then screening their F1 progeny for transgenic or mutant larvae. However, transgenesis in S. stercoralis has been severely hindered by the inability to establish stable transgenic lines that can be propagated for multiple generations through a host; to date, studies of transgenic S. stercoralis have been limited to heterogeneous populations of transgenic F1 larvae. Here, we develop an efficient pipeline for the generation of stable transgenic lines in S. stercoralis. We also show that this approach can be used to efficiently generate stable transgenic lines in the rat-infective nematode Strongyloides ratti. The ability to generate stable transgenic lines circumvents the limitations of working with heterogeneous F1 populations, such as variable transgene expression and the inability to generate transgenics of all life stages. Our transgenesis approach will enable novel lines of inquiry into parasite biology, such as transgene-based comparisons between free-living and parasitic generations.

Introduction to Strongyloides stercoralis Anatomy.

Strongyloides stercoralis, commonly known as the human threadworm, is a skin-penetrating gastrointestinal parasitic nematode that infects hundreds of millions of people worldwide. Like other Strongyloides species, S. stercoralis is capable of cycling through a single free-living generation. Although S. stercoralis and the free-living nematode Caenorhabditis elegans are evolutionarily distant, the free-living adults of S. stercoralis are similar enough in size and morphology to C. elegans adults that techniques for generating transgenics and knockouts in C. elegans have been successfully adapted for use in S. stercoralis. High-quality genomic and transcriptomic data are also available for S. stercoralis. Thus, one can use a burgeoning array of functional genomic tools in S. stercoralis to probe questions about parasitic nematode development, physiology, and behavior. Knowledge gained from S. stercoralis will inform studies of other parasitic nematodes such as hookworms that are not yet amenable to genetic manipulation. This review describes the basic anatomy of S. stercoralis.

Skin-penetrating nematodes exhibit life-stage-specific interactions with host-associated and environmental bacteria

BackgroundSkin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points during their life cycle, Strongyloides species encounter a wide variety of host-associated and environmental bacteria. However, the microbiome associated with Strongyloides species, and the behavioral and physiological interactions between Strongyloides species and bacteria, remain unclear.ResultsWe first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have an associated microbiome consisting of specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to a narrow range of environmental bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, and thereby greatly decreases parasite viability.ConclusionsSkin-penetrating nematodes encounter bacteria from various ecological niches throughout their life cycle. Our results demonstrate that bacteria function as key chemosensory cues for directing parasite movement in a life-stage-specific manner. Some bacterial genera may form essential associations with the nematodes, while others are detrimental and serve as a potential source of novel nematicides.

Клинические случаи стронгилоидоза кошек и собак в Московском регионе

Carnivores’, monkeys’ and humans’ Strongyloidosis is caused by nematodes Strongyloides stercoralis. Helminths are localized in the small (rarely ― thick) part of the intestine. Invasion in carnivores and humans is more common in regions with warm and humid climates, but recently, cases of its registration in countries with moderate and even cold climates are observed. The feature of strongylatosis infestation is the presence of 2 helminth’s generations ― parasitic and free-living, herewith species of free-living generations remain and develop in the external environment, including premises, but parasitic species infect animals by oral or percutaneous ways, as well as cause auto-infestation. Clinically strongyloidosis is manifested in young animals, as well as in animals in immunodeficiency states. Nematodes injure the intestinal mucosa, causing inflammation, swelling, and erosion. As a result, the of nutrients absorption’s and assimilation’s process is disrupted. Signs of catarrhal enteritis are clinically observed― diarrhea (often with blood), anorexia, slowing growth and development. During the migration of larvae through the lungs, bronchopneumonia is observed, including the addition of secondary bacterial infections; with percutaneous infection ― itching and dermatitis. For the diagnosis of the infestation is preferable to use helmintological methods (Berman’s method). Against Strongyloides the efficiency of benzimidazoles, avermectins and milbemycin is noticed. The authors describe cases of detection of nematodes Strongyloides stercoralis in cats and dogs in Moscow region. For deworming of animals was used a combination of 2 anthelmintic with a synergistic effect against adult stages and larvae of strongyloidiasis: ivermectin and fenbendazole.

Description, molecular characterization and life cycle of Serpentirhabdias mussuranae n. sp. (Nematoda: Rhabdiasidae) from Clelia clelia (Reptilia: Colubroidea) in Brazil.

Serpentirhabdias mussuranae n. sp. is described from the lungs of the mussurana, Clelia clelia (Daudin, 1803), from vicinities of Lábrea, Amazonas State, Brazil. The species is characterized by the triangular oral opening, the presence of teeth (onchia) in the oesophastome, the excretory glands longer than the oesophagus and the tail abruptly narrowing in its anterior half and gradually tapering in posterior half. Among the Neotropical representatives of the genus, three species are known to possess the onchia in the oesophastome: S. atroxi, S. moi and S. viperidicus. Serpentirhabdias mussuranae n. sp. differs from S. atroxi and S. viperidicus by its triangular shape of the oral opening and the oesophastome in apical view, vs. round in the latter two congeners. Additionally, S. viperidicus has a larger oesophastome, 13-22 micrometers wide and 13-23 micrometers deep. The new species has relatively longer excretory glands than S. moi. The new species is morphologically and genetically close to S. atroxi, S. moi and S. viperidicus, all parasitic in Brazilian snakes, based on the presence of onchia and the comparison of nucleotide sequences of nuclear ribosomal DNA and mitochondrial cox1 gene (differences varied between 3.8% and 7.1%). Data on the life cycle of S. mussuranae n. sp. is provided, and the life cycle is typical of the genus Serpentirhabdias, with the combination of direct development and heterogony. Free-living larval stages and the adults of amphimictic free-living generation are described. The results of molecular phylogenetic analysis based on nuclear ribosomal internal transcribed spacer (ITS) + partial 28S region and partial mitochondrial cox1 gene are provided.

Morphological features of development of Strongyloides westeri (Nematoda, Rhabditida) in vitro

Strongyloides westeri (Ihle, 1917), a parasitic horse nematode, has an unusual lifecycle, which allows it to exist for a long time in the environment. Morphometric features of eggs, larvae and free-living S. westeri were studied in vitro under different temperature regimes. The optimal temperature for their embryonic development is 25 °С, under which 90% of the first stage rhabditiform larvae are formed and released within 7 hours of cultivation. The temperatures of 20 and 30 °С are less favorable for their development. Embryonic development of Strongyloides has four stages that differ in morphology and size. The eggs of a parthenogenetic female are 3.7% longer and 19.6% wider than eggs isolated from free-living females of S. westeri. In embryogenesis, the eggs shorten by 4.4 μm (6.5%) and widen by 5.35 μm (8.3%). New data were obtained on postembryonic development of S. westeri. The differential morphometric features of stage 1 and 2 rhabditiform larvae which grow both in length and width (33.7% and 30.4% respectively) are established. The development of filariform larvae is associated with loss of bulbous thickening and formation of cylindrical oesophagus. Simultaneously, the body elongates, and the gut becomes shorter. Differential morphometric features of free-living males and females of S. westeri are the length and width of body, length of oesophagus, gut, tail end, and size of spicules. Postembryonic development of the free-living and parasitic generations from rhabditiform larvae is temperature-dependent. Most of the free-living generations of Strongyloides (54.0%) are formed at 20 °С, and filariform larvae mostly (70.0%) develop at 30 °С. The obtained results of morphological studies improve differential diagnostics of the nematode at various stages of development and further advance the study of its intraspecific variability.

Gain and Loss of Small RNA Classes-Characterization of Small RNAs in the Parasitic Nematode Family Strongyloididae.

The nematode family Strongyloididae is of particular interest because it contains important parasites of medical and veterinary relevance. In addition, species of this family can form parasitic and free-living generations and it also occupies an interesting phylogenetic position within the nematodes. Nematodes differ in several ways from other taxa with respect to their small noncoding RNAs. Recent comparative studies revealed that there is also considerable variability within the nematodes. However, no Strongyloididae species or close relative was included in these studies. We characterized the small RNAs of two developmental stages of three different Strongyloididae species and compared them with the well-studied free-living nematodes Caenorhabditis elegans and Pristionchus pacificus. Strongyloididae have conserved and taxon-specific microRNAs, many of which are differentially regulated between the two developmental stages. We identified a novel class of around 27-nucleotide-long RNAs starting with 5′G or A, of which a large fraction have the potential to target transposable elements. These RNAs most likely have triphosphates at their 5′ ends and are therefore presumably synthesized by RNA-dependent RNA polymerases. In contrast to C. elegans but similarly to some other nematode taxa, Strongyloididae have no Piwi-interacting RNAs, nor do their genomes encode Argonaute proteins of the Piwi family. Finally, we attempted but failed to detect circulating parasite small RNAs in the blood of hosts.

Different but overlapping populations of Strongyloides stercoralis in dogs and humans-Dogs as a possible source for zoonotic strongyloidiasis.

Strongyloidiasis is a much-neglected soil born helminthiasis caused by the nematode Strongyloides stercoralis. Human derived S. stercoralis can be maintained in dogs in the laboratory and this parasite has been reported to also occur in dogs in the wild. Some authors have considered strongyloidiasis a zoonotic disease while others have argued that the two hosts carry host specialized populations of S. stercoralis and that dogs play a minor role, if any, as a reservoir for zoonotic S. stercoralis infections of humans. We isolated S. stercoralis from humans and their dogs in rural villages in northern Cambodia, a region with a high incidence of strongyloidiasis, and compared the worms derived from these two host species using nuclear and mitochondrial DNA sequence polymorphisms. We found that in dogs there exist two populations of S. stercoralis, which are clearly separated from each other genetically based on the nuclear 18S rDNA, the mitochondrial cox1 locus and whole genome sequence. One population, to which the majority of the worms belong, appears to be restricted to dogs. The other population is indistinguishable from the population of S. stercoralis isolated from humans. Consistent with earlier studies, we found multiple sequence variants of the hypervariable region I of the 18 S rDNA in S. stercoralis from humans. However, comparison of mitochondrial sequences and whole genome analysis suggest that these different 18S variants do not represent multiple genetically isolated subpopulations among the worms isolated from humans. We also investigated the mode of reproduction of the free-living generations of laboratory and wild isolates of S. stercoralis. Contrary to earlier literature on S. stercoralis but similar to other species of Strongyloides, we found clear evidence of sexual reproduction. Overall, our results show that dogs carry two populations, possibly different species of Strongyloides. One population appears to be dog specific but the other one is shared with humans. This argues for the strong potential of dogs as reservoirs for zoonotic transmission of S. stercoralis to humans and suggests that in order to reduce the exposure of humans to infective S. stercoralis larvae, dogs should be treated for the infection along with their owners.

Parasitological and transcriptomic comparison of Strongyloides ratti infections in natural and in suboptimal permissive hosts

The nematode genus Strongyloides consists of fairly species-specific small intestinal parasites of various vertebrates, among them the human pathogen S. stercoralis. Between the parthenogenetic parasitic generations these worms can also form single facultative sexual free-living generations. In addition to their primary hosts, several species can also live more or less well in other permissive hosts, which are sometimes not very closely related with the normal host. For example, S. stercoralis can also infect dogs and non-human primates.Here we compare the infection and reproductive success over time and the gene expression profiles as determined by quantitative sequencing of S. ratti parasitizing in its natural host rat and in the permissive host gerbil. We show that in gerbils fewer infective larvae successfully establish in the host, but those that do accomplish this survive and reproduce for longer and produced a higher proportion of males during the first two month of infection. Globally, the gene expression profiles in the two hosts are very similar. Among the relatively few differentially expressed genes, astacin-like and acetylcholinesterase genes are prominently represented. In the future it will be interesting to see if these changes in the suboptimal host are indeed ecologically sensible responses to the different host.

Optimizing culture conditions for free-living stages of the nematode parasite Strongyloides ratti

The rat parasitic nematode Strongyloides ratti (S. ratti) has recently emerged as a model system for various aspects of parasite biology and evolution. In addition to parasitic parthenogenetic females, this species can also form facultative free-living generations of sexually reproducing adults. These free-living worms are bacteriovorous and grow very well when cultured in the feces of their host. However, in fecal cultures the worms are rather difficult to find for observation and experimental manipulation. Therefore, it has also been attempted to raise S. ratti on Nematode Growth Media (NGM) plates with Escherichia coli OP50 as food, exactly as described for the model nematode Caenorhabditis elegans.Whilst worms did grow on these plates, their longevity and reproductive output compared to fecal cultures were dramatically reduced. In order to improve the culture success we tested other plates occasionally used for C. elegans and, starting from the best performing one, systematically varied the plate composition, the temperature and the food in order to further optimize the conditions. Here we present a plate culturing protocol for free-living stages of S. ratti with strongly improved reproductive success and longevity.

Strongyloides ratti and S. venezuelensis - rodent models of Strongyloides infection.

Strongyloides spp. are common parasites of vertebrates and two species, S. ratti and S. venezuelensis, parasitize rats; there are no known species that naturally infect mice. Strongyloides ratti and S. venezuelensis overlap in their geographical range and in these regions co-infections appear to be common. These species have been widely used as tractable laboratory systems in rats as well as mice. The core biology of these two species is similar, but there are clear differences in aspects of their within-host biology as well as in their free-living generation. Phylogenetic evidence suggests that S. ratti and S. venezuelensis are the result of two independent evolutionary transitions to parasitism of rats, which therefore presents an ideal opportunity to begin to investigate the basis of host specificity in Strongyloides spp.

Superproof about Sexual Reproduction and Life Cycle in the Parasitic Generation of Strongyloides stercoralis in Human Host

Background and study aim: Strongyloides stercoralis was believed to have two kinds of life cycles. One of them was an internal parthenogenetic life cycle that constituted the parasitic generation. Some authors also stated that this parasitic generation of the internal life cycle used to reproduce only by protandrogony (i.e., hermaphroditism). The second type of life cycle was the external sexual cycle, interacting among free-living worms that represented the free-living generation in soil. The key objectives of this study were to: verify the type of reproduction in the parasitic generation of S. stercoralis, and demonstrate the complete integrated life cycles of both parasitic and free-living generations of S. stercoralis. Patients and Methods: The major sites of stool sample collection were selected to be the elementary schools (from students) at Dilla District, because S. stercoralis was ascertained to be endemic in this region. The parasitic worms of Strongyloides stercoralis obtained from fresh stools were used to set growth culture of free-living generation of this very species and to find the free-living male and female in copulation from the growth culture. A set of parasitic male & female in copulation isolated from a fresh stool sample of a patient under medical care in a hospital was also included. Results: The male and female worms of S. stercoralis had been isolated from both parasitic and free-living generations while they were in the actual copulation. Conclusion: The method of reproduction in the parasitic generation of S. stercoralis in human host was practically proved to be certainly sexual. This verified conclusion was the first practical achievement in the entire globe by solving the persistent problems (i.e., erroneous concepts of parthenogenesis and protandrogony as the types of reproduction in the parasitic generation of S. stercoralis) that had been misleading and puzzling the minds of the concerned scientists of the world until the establishment of this very achievement. Additionally, this achievement had enabled the investigator to construct the complete integrated life cycles of both parasitic and free-living generations of S. stercoralis.

The morphology of free-living stages and immature parasites of Rhabdias paraensis (Nematoda: Rhabdiasidae), a parasite of Rhinella marina (Anura: Bufonidae) in Brazil.

Rhabdias paraensis Santos, Melo, Nascimento, Nascimento, Giese et Furtado, 2011 was described based on fully gravid worms. Further investigations on the free-living stages, immature worms and young individuals were facilitated by cultivation in the laboratory, which allowed us to add new information about the morphology and development of the species. Observations on the free-living development of R. paraensis showed that the life cycle is typical of Rhabdias, with alternation of gonochoristic and hermaphroditic generations and without homogony. Males of the free-living generation were different from those in several species of the genus studied previously. In the original description, the excretory glands and duct were absent in gravid specimens of R. paraensis, while in this study, distinct excretory glands and a duct were observed in immature and young individuals. Additionally, we recognised the separation of the buccal capsule walls into anterior and posterior portions and described the specific shapes of these portions in lateral and apical view. Studies on the morphology and development of free-living stages of Rhabdias spp. from Neotropical regions may provide additional information for species determination.

Differential chromatin amplification and chromosome complements in the germline of Strongyloididae (Nematoda).

Nematodes of the genus Strongyloides are intestinal parasites of vertebrates including man. Currently, Strongyloides and its sister genus Parastrongyloides are being developed as models for translational and basic biological research. Strongyloides spp. alternate between parthenogenetic parasitic and single free-living sexual generations, with the latter giving rise to all female parasitic progeny. Parastrongyloides trichosuri always reproduces sexually and may form many consecutive free-living generations. Although the free-living adults of both these species share a superficial similarity in overall appearance when compared to Caenorhabditis elegans, there are dramatic differences between them, in particular with respect to the organization of the germline. Here we address two such differences, which have puzzled investigators for several generations. First, we characterize a population of non-dividing giant nuclei in the distal gonad, the region that in C. elegans is populated by mitotically dividing germline stem cells and early meiotic cells. We show that in these nuclei, autosomes are present in higher copy numbers than X chromosomes. Consistently, autosomal genes are expressed at higher levels than X chromosomal ones, suggesting that these worms use differential chromatin amplification for controlling gene expression. Second, we address the lack of males in the progeny of free-living Strongyloides spp. We find that male-determining (nullo-X) sperm are present in P. trichosuri, a species known to produce male progeny, and absent in Strongyloides papillosus, which is consistent for a species that does not. Surprisingly, nullo-X sperm appears to be present in Strongyloides ratti, even though this species does not produce male progeny. This suggests that different species of Strongyloides employ various strategies to prevent the formation of males in the all-parasitic progeny of the free-living generation.

Strongyloides stercoralis and Relatives: Recent Advances in General and Molecular Biology

Human strongyloidiasis is a threat to global health, presenting significant challenges in diagnosis and clinical management. The imperative to incorporate strongyoidiasis more fully into control programs for soil-transmitted helminths is increasingly recognized. The unique life cycles of S. stercoralis and congeneric species contain both free-living and parasitic generations, and transcriptomic methods have recently identified genes of potential importance to parasitism in these parasites. Proteomics recently revealed stage-specific secreted proteins that appear crucial to the host-parasite interaction. A comprehensive genome sequencing project for Strongyloides spp. is now nearing completion. Recent technical advances in transgenesis for S. stercoralis and S. ratti, including the first establishment of stable transgenic lines, promise to advance functional evaluations of genes expressed in conjunction with crucial life cycle events. Studies employing these methods recently bolstered the hypothesis that S. stercoralis uses cellular signaling pathways homologous to three that regulate dauer larval development in Caenorhabditis elegans to regulate morphogenesis and development of its infective third-stage larva. The free-living generation of Strongyloides makes classical genetics formally possible. Recent advances, such as a genetic map of S. ratti and a molecular genetic and karyotypic analysis of sex determination in S. papillosus, will greatly facilitate this approach. Advanced methods for study of chemosensation in C. elegans were recently applied to discover numerous host attractant molecules that mediate host finding and contact by infective third-stage larvae of Strongyloides spp. Finally, nucleic acid-based diagnostic methods have recently come to the fore as alternatives to parasitological and immunodiagnostic techniques.

How to become a parasite without sex chromosomes: a hypothesis for the evolution of Strongyloides spp. and related nematodes.

Parasitic lifestyles evolved many times independently. Just within the phylum Nematoda animal parasitism must have arisen at least four times. Switching to a parasitic lifestyle is expected to lead to changes in various life history traits including reproductive strategies. Parasitic nematode worms of the genus Strongyloides represent an interesting example to study these processes because they are still capable of forming facultative free-living generations in between parasitic ones. The parasitic generation consists of females only, which reproduce parthenogenetically. The sex in the progeny of the parasitic worms is determined by environmental cues, which control a, presumably ancestral, XX/XO chromosomal sex determining system. In some species the X chromosome is fused with an autosome and one copy of the X-derived sequences is removed by sex-specific chromatin diminution in males. Here I propose a hypothesis for how today's Strongyloides sp. might have evolved from a sexual free-living ancestor through dauer larvae forming free-living and facultative parasitic intermediate stages.

A survey of the fern gametophyte flora of Japan: Frequent independent occurrences of noncordiform gametophytes

Ferns and lycophytes are the only extant land plants with two free-living generations (sporophytes and gametophytes); hence, a single species may have two different distributions. The distribution of the gametophytes of most fern species, which are much smaller in size than sporophytes, are almost unknown due to the difficulty of identifying gametophytes using morphological characters. Twelve quadrats (1 m(2) or 0.25 m(2)), each subdivided into a grid of 100 (10 × 10) or 25 (5 × 5) squares, were used to survey gametophytes in the Japanese Archipelago, where distribution data of sporophytes and "DNA barcodes" for identification of gametophytes have fully been established in previous studies. Collected gametophytes were identified using the plastid rbcL-a region. In total, gametophytes of 38 species in two broad morphological categories (28 cordiform and 10 noncordiform species) were identified among 407 collections. The cordiform gametophytes discovered are without exception accompanied by their conspecific sporophytes at the periphery of the quadrats. On the other hand, the sporophytic counterparts of the noncordiform gametophytes are often not found or are rare around the sites. This study demonstrates with a regional flora that fern gametophytes do not always co-occur with sporophytes of the same species. In particular, noncordiform gametophytes tended to occur independently of conspecific sporophytes. This pattern may be due to the capability for indeterminate growth and vegetative reproduction by gemmae in noncordiform gametophytes.

Life Cycle Stage-resolved Proteomic Analysis of the Excretome/Secretome from Strongyloides ratti—Identification of Stage-specific Proteases

A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.

A genetic map of the animal-parasitic nematode Strongyloides ratti

Classical genetic approaches are rarely used with metazoan endo-parasites, largely because the adult stages are usually hidden within hosts, making controlled crosses difficult. The nematode Strongyloides ratti is a parasite of the small intestine of rats, and is a relative of the parasite of humans S. stercoralis. The life-cycle of Strongyloides spp. has a facultative free-living adult generation. Here we describe procedures for genetic mapping, and a genetic map, for S. ratti. This is, as far as we are aware, the first genetic map of an animal parasitic nematode. This significantly improves the usefulness of S. ratti as experimentally tractable system for parasitological investigations and for comparative studies with the model nematode Caenorhabditis elegans.

Molecular and functional characterisation of the heat shock protein 10 of Strongyloides ratti

Strongyloides stercoralis and S. ratti are intestinal parasitic nematodes infecting rats and humans, respectively. Both present extraordinary life cycles comprising a free-living generation in addition to parasitic stages. In search of molecules possibly involved in parasite–host interaction, we performed mass spectrometry to identify excretory/secretory products of S. ratti. Amongst others we detected homologs of the heat shock proteins HSP10 and HSP60 (Sr-HSP10 and Sr-HSP60). HSPs are well known as chaperones involved in stress responses of cells, but recent studies suggest additional roles of small HSPs for parasite biology including immune modulation. To characterise Sr-HSP10, we cloned its full-length cDNA, analysed the genomic organisation, tested its presumptive role as an interaction partner of Sr-HSP60, studied its transcription in the parasite, and expressed the protein to test its immune responses. The cDNA contains an open reading frame of 330bp encoding a polypeptide of 110 amino acids with an approximate molecular weight of 10kDa. The Sr-HSP10 protein is highly homologous to that of the human pathogen S. stercoralis with only eight amino acid substitutions. Analysis of the genomic organisation of the Sr-HSP10 locus revealed that the gene is linked head-to-head to the gene encoding Sr-HSP60, and both share a bidirectional promoter. RT-PCR experiments indicated potential independent expression of the Sr-HSPs genes. In situ hybridisation results demonstrate Sr-HSP10 transcription in the gut area. Mammalian and yeast two-hybrid assays show dimerisation of Sr-HSP10, but no binding to recombinant Sr-HSP60. Immunisation experiments finally revealed a strong immunogenicity of Sr-HSP10 and provided evidence for a role in regulating the host–parasite interaction.