The parasitic nematode Strongyloides ratti exists predominantly as populations of long-lived asexual lineages.

Nematodes are important parasites of people and animals, and in natural ecosystems they are a major ecological force. Strongyloides ratti is a common parasitic nematode of wild rats and we have investigated its population genetics using single-worm, whole-genome sequencing. We find that S. ratti populations in the UK consist of mixtures of mainly asexual lineages that are widely dispersed across a host population. These parasite lineages are likely very old and may have originated in Asia from where rats originated. Genes that underly the parasitic phase of the parasite's life cycle are hyperdiverse compared with the rest of the genome, and this may allow the parasites to maximise their fitness in a diverse host population. These patterns of parasitic nematode population genetics have not been found before and may also apply to Strongyloides spp. that infect people, which will affect how we should approach their control.

A comparative 'omics' approach for prediction of candidate Strongyloides stercoralis diagnostic coproantigens.

Human infection with the intestinal nematode Strongyloides stercoralis is persistent unless effectively treated, and potentially fatal in immunosuppressed individuals. Epidemiological data are lacking, partially due to inadequate diagnosis. A rapid antigen detection test is a priority for population surveillance, validating cure after treatment, and for screening prior to immunosuppression. We used a targeted analysis of open access 'omics' data sets and used online predictors to identify S. stercoralis proteins that are predicted to be present in infected stool, Strongyloides-specific, and antigenic. Transcriptomic data from gut and non-gut dwelling life cycle stages of S. stercoralis revealed 328 proteins that are differentially expressed. Strongyloides ratti proteomic data for excreted and secreted (E/S) proteins were matched to S. stercoralis, giving 1,057 orthologues. Five parasitism-associated protein families (SCP/TAPS, prolyl oligopeptidase, transthyretin-like, aspartic peptidase, acetylcholinesterase) were compared phylogenetically between S. stercoralis and outgroups, and proteins with least homology to the outgroups were selected. Proteins that overlapped between the transcriptomic and proteomic datasets were analysed by multiple sequence alignment, epitope prediction and 3D structure modelling to reveal S. stercoralis candidate peptide/protein coproantigens. We describe 22 candidates from seven genes, across all five protein families for further investigation as potential S. stercoralis diagnostic coproantigens, identified using open access data and freely-available protein analysis tools. This powerful approach can be applied to many parasitic infections with 'omic' data to accelerate development of specific diagnostic assays for laboratory or point-of-care field application.

RNAi-mediated knockdown of daf-12 in the model parasitic nematode Strongyloides ratti.

The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.

Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite Strongyloides ratti.

Infections with helminth parasites are controlled by a concerted action of innate and adaptive effector cells in the frame of a type 2 immune response. Basophils are innate effector cells that may also contribute to the initiation and amplification of adaptive immune responses. Here, we use constitutively basophil-deficient Mcpt8-Cre mice to analyze the impact of basophils during initiation and execution of the protective type 2 responses to both, a primary infection and a challenge infection of immune mice with the helminth parasite Strongyloides ratti. Basophil numbers expanded during parasite infection in blood and mesenteric lymph nodes. Basophil deficiency significantly elevated intestinal parasite numbers and fecal release of eggs and larvae during a primary infection. However, basophils were neither required for the initiation of a S. ratti-specific cellular and humoral type 2 immune response nor for the efficient protection against a challenge infection. Production of Th2 cytokines, IgG1 and IgE as well as mast cell activation were not reduced in basophil-deficient Mcpt8-Cre mice compared to basophil-competent Mcpt8-WT littermates. In addition, a challenge infection of immune basophil-deficient and WT mice resulted in a comparable reduction of tissue migrating larvae, parasites in the intestine and fecal release of eggs and L1 compared to mice infected for the first time. We have shown previously that S. ratti infection induced expansion of Foxp3+ regulatory T cells that interfered with efficient parasite expulsion. Here we show that depletion of regulatory T cells reduced intestinal parasite burden also in absence of basophils. Thus basophils were not targeted specifically by S. ratti-mediated immune evasive mechanisms. Our collective data rather suggests that basophils are non-redundant innate effector cells during murine Strongyloides infections that contribute to the early control of intestinal parasite burden.

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Targeted mutagenesis in a human-parasitic nematode.

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

Th2/1 Hybrid Cells Occurring in Murine and Human Strongyloidiasis Share Effector Functions of Th1 Cells.

Infections by the soil-transmitted threadworm Strongyloides stercoralis affect 30-100 million people worldwide, predominantly in tropic and sub-tropic regions. Here we assessed the T helper cell phenotypes in threadworm-infected patients and experimental murine infections with focus on CD4+ T cells co-expressing markers of Th2 and Th1 differentiation. We show that mice infected with the close relative S. ratti generate strong Th2 responses characterized by the expansion of CD4+ GATA-3+ cells expressing IL-4/-5/-13 in blood, spleen, gut-draining lymph nodes, lung and gut tissue. In addition to conventional Th2 cells, significantly increased frequencies of GATA-3+T-bet+ Th2/1-hybrid cells were detected in all organs and co-expressed Th2- and Th1-cytokines at intermediate levels. Assessing the phenotype of blood-derived CD4+ T cells from South Indian patients infected with S. stercoralis and local uninfected control donors we found that GATA-3 expressing Th2 cells were significantly increased in the patient cohort, coinciding with elevated eosinophil and IgE/IgG4 levels. A fraction of IL-4+CD4+ T cells simultaneously expressed IFN-γ hence displaying a Th2/1 hybrid phenotype. In accordance with murine Th2/1 cells, human Th2/1 cells expressed intermediate levels of Th2 cytokines. Contrasting their murine counterparts, human Th2/1 hybrids were marked by high levels of IFN-γ and rather low GATA-3 expression. Assessing the effector function of murine Th2/1 cells in vitro we found that Th2/1 cells were qualified for driving the classical activation of macrophages. Furthermore, Th2/1 cells shared innate, cytokine-driven effector functions with Th1 cells. Hence, the key findings of our study are that T helper cells with combined characteristics of Th2 and Th1 cells are integral to immune responses of helminth-infected mice, but also occur in helminth-infected humans and we suggest that Th2/1 cells are poised for the instruction of balanced immune responses during nematode infections.

Duplications and Positive Selection Drive the Evolution of Parasitism-Associated Gene Families in the Nematode Strongyloides papillosus.

Gene duplication is a major mechanism playing a role in the evolution of phenotypic complexity and in the generation of novel traits. By comparing parasitic and nonparasitic nematodes, a recent study found that the evolution of parasitism in Strongyloididae is associated with a large expansion in the Astacin and CAP gene families.To gain novel insights into the developmental processes in the sheep parasite Strongyloides papillosus, we sequenced transcriptomes of different developmental stages and sexes. Overall, we found that the majority of genes are developmentally regulated and have one-to-one orthologs in the diverged S. ratti genome. Together with the finding of similar expression profiles between S. papillosus and S. ratti, these results indicate a strong evolutionary constraint acting against change at sequence and expression levels. However, the comparison between parasitic and free-living females demonstrates a quite divergent pattern that is mostly due to the previously mentioned expansion in the Astacin and CAP gene families. More detailed phylogenetic analysis of both gene families shows that most members date back to single expansion events early in the Strongyloides lineage and have undergone subfunctionalization resulting in clusters that are highly expressed either in infective larvae or in parasitic females. Finally, we found increased evidence for positive selection in both gene families relative to the genome-wide expectation.In summary, our study reveals first insights into the developmental transcriptomes of S. papillosus and provides a detailed analysis of sequence and expression evolution in parasitism-associated gene families.

A protocol for chemical mutagenesis in Strongyloides ratti.

Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing.

Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes.

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

Characterization of genes with a putative key role in the parasitic lifestyle of the nematode Strongyloides ratti.

Parasitic nematodes are significant pathogens of humans and other animals. The molecular and genetic basis of animal parasitism is not yet fully understood. Strongyloides spp. are a genus of gastrointestinal nematodes of which species infect approximately 100­200 million people worldwide. S. ratti is a natural parasite of the rat, and a useful and amenable laboratory model. Previous EST and microarray analyses of the S. ratti life cycle have identified genes whose expression was specific, or biased, to the parasitic adult stage, suggesting that they may play a key role in parasitism in this species. Here we have further investigated the expression of these genes (by RT-PCR) throughout the S. ratti life-cycle. We produced recombinant proteins in vitro for a subset of these genes, which were used in Western blot analyses to investigate the distribution of the gene products among different stages of the S. ratti life cycle. We tested the efficacy of these recombinant proteins as anti-S. ratti vaccines. One of the proteins was detected in the excretory/secretory products of the parasitic stages.

Characterization of a secreted macrophage migration inhibitory factor homologue of the parasitic nematode Strongyloides acting at the parasite-host cell interface.

Strongyloidiasis is a tropical parasitosis characterized by an alternation between free-living and parasitic stages, and by long-term infection via autoinfection. Since invasion and evasion processes of helminth parasites are substantially attained by the involvement of excretory-secretory products, we identified and characterized the 13.5 kDa macrophage migration inhibitory factor (MIF)-like protein in Strongyloides ratti. Sra-MIF is mainly secreted from the infective stage larvae (iL3), while the transcript was found at lower levels in parasitic and free-living females. Sequence analysis of the full-length cDNA showed the highest homology to the human pathogen Strongyloides stercoralis, and both are related to the MIF type-2. Unlike other mif genes, the Sra-mif includes no intron. The protein was recombinantly expressed in Escherichia coli and purified. Sra-MIF exhibited no in vitro tautomerase activity. The exposure of Sra-MIF to the host immune system is confirmed by high IgG reactivities found in the hosts' sera following infection or immunization. Flow cytometric analysis indicated the binding of Sra-MIF to the monocytes/macrophage lineage but not to peripheral lymphocytes. After exposure to Sra-MIF, monocytes released IL-10 but not TNF-alpha suggesting the involvement of the secreted parasite MIF in host immune responses.

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes.

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.

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.

Transgenesis in the parasitic nematode Strongyloides ratti.

Strongyloides and related genera are advantageous subjects for transgenesis in parasitic nematodes, primarily by gonadal microinjection as has been used with Caenorhabditis elegans. Transgenesis has been achieved in Strongyloides stercoralis and in Parastrongyloides trichosuri, but both of these lack well-adapted, conventional laboratory hosts in which to derive transgenic lines. By contrast, Strongyloides ratti develops in laboratory rats with high efficiency and offers the added advantages of robust genomic and transcriptomic databases and substantial volumes of genetic, developmental and immunological data. Therefore, we evaluated methodology for transgenesis in S. stercoralis as a means of transforming S. ratti. S. stercoralis-based GFP reporter constructs were expressed in a proportion of F1 transgenic S. ratti following gonadal microinjection into parental free-living females. Frequencies of transgene expression in S. ratti, ranged from 3.7% for pAJ09 to 6.8% for pAJ20; respective frequencies for these constructs in S. stercoralis were 5.6% and 33.5%. Anatomical patterns of transgene expression were virtually identical in S. ratti and S. stercoralis. This is the first report of transgenesis in S. ratti, an important model organism for biological investigations of parasitic nematodes. Availability of the rat as a well-adapted laboratory host will facilitate derivation of transgenic lines of this parasite.

Response of the Strongyloides ratti transcriptome to host immunological environment.

The immunological environment experienced by parasitic nematodes varies greatly between hosts and is particularly influenced by whether or not a host has been previously infected. How a parasitic nematode responds to these different environments is poorly understood, but may allow a parasite to ameliorate the adverse effects of host immunity on parasite fitness. Here we use a microarray approach to identify genes in the parasitic nematode Strongyloides ratti that exhibit differential transcription between different rat host immunological environments, and between replicate lines of S. ratti selected for either early or late reproduction. We hypothesise that such genes may be used by this species to cope with and respond to its host environment. Our results showed that, despite large phenotypic differences between S. ratti adults from different immunological environments, the S. ratti transcriptome exhibited a relatively stable pattern of expression. Thus, differential expression amongst treatments was limited to a small proportion of transcripts and generally involved only modest fold changes. These transcripts included a group of collagen genes up-regulated in parasites early in an infection, and in immunised host environments, which may be related to protection against the damage caused to a parasite by host immune responses. We found that later in an infection, a number of genes associated with muscle function and repair were up-regulated in immunised host environments; these may help parasites maintain their position in the host intestine. Differences in transcription between selection lines of S. ratti were only observed in immunised hosts and included genes associated with the response to the host's immunological environment.

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.

Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure.

BACKGROUND: Aspartic proteases are known to play an important role in the biology of nematode parasitism. This role is best characterised in blood-feeding nematodes, where they digest haemoglobin, but they are also likely to play important roles in the biology of nematode parasites that do not feed on blood. In the present work, we investigate the evolution and expression of aspartic proteases in Strongyloides ratti, which permits a unique comparison between parasitic and free-living adult forms within its life-cycle. RESULTS: We identified eight transcribed aspartic protease sequences and a further two genomic sequences and compared these to homologues in Caenorhabditis elegans and other nematode species. Phylogenetic analysis demonstrated a complex pattern of gene evolution, such that some S. ratti sequences had a one-to-one correspondence with orthologues of C. elegans but that lineage-specific expansions have occurred for other aspartic proteases in these two nematodes. These gene duplication events may have contributed to the adaptation of the two species to their different lifestyles. Among the set of S. ratti aspartic proteases were two closely-related isoforms that showed differential expression during different life stages: ASP-2A is highly expressed in parasitic females while ASP-2B is predominantly found in free-living adults. Molecular modelling of the ASP-2 isoforms reveals that their substrate specificities are likely to be very similar, but that ASP-2B is more electrostatically negative over its entire molecular surface than ASP-2A. This characteristic may be related to different pH values of the environments in which these two isoforms operate. CONCLUSIONS: We have demonstrated that S. ratti provides a powerful model to explore the genetic adaptations associated with parasitic versus free-living life-styles. We have discovered gene duplication of aspartic protease genes in Strongyloides and identified a pair of paralogues differentially expressed in either the parasitic or the free-living phase of the nematode life-cycle, consistent with an adaptive role for aspartic proteases in the evolution of nematode parasitism.

Transcript profiles of long- and short-lived adults implicate protein synthesis in evolved differences in ageing in the nematode Strongyloides ratti.

The nematode Strongyloides ratti shows remarkable phenotypic plasticity in ageing, with parasitic adults living at least 80-times longer than free-living adults. Given that long- and short-lived adults are genetically identical, this plasticity is likely to be due to differences in gene expression. To try and understand how this inter-morph difference in longevity evolved, we compared gene expression in long- and short-lived adults. DNA microarray analysis of long- and short-lived adults identified 32 genes that were up-regulated in long-lived adults, and 96 genes up-regulated in short-lived adults. Strikingly, 38.5% of the genes expressed more in the short-lived morph are predicted to encode ribosomal proteins, compared with only 9% in the long-lived morph. Among the 32 longevity-associated genes there was very little enrichment of genes linked to cellular maintenance. Overall, we have therefore observed a negative correlation between expression of ribosomal protein genes and longevity in S. ratti. Interestingly, engineered reduction of expression of ribosomal protein genes increases lifespan in the free-living nematode Caenorhabditis elegans. Our study therefore suggests that differences in levels of protein synthesis could contribute to evolved differences in animal longevity.