Sex determination: a worm does it by elimination

Parasitic nematode worms of the genus Strongyloides have an alternation of many asexual, all-female generations with a sexual generation composed of males and females. Males of S. papillosus have now been shown to be produced by elimination of chromosomal material that constitutes the X chromosome in its close relatives.     

Sex determination in the parasitic nematode Strongyloides ratti

The parasitic nematode Strongyloides ratti reproduces by both parthenogenesis and sexual reproduction, but its genetics are poorly understood. Cytological evidence suggests that sex determination is an XX/XO system. To investigate this genetically, we isolated a number of sex-linked DNA markers. One of these markers, Sr-mvP1, was shown to be single copy and present at a higher dose in free-living females than in free-living males. The inheritance of two alleles of Sr-mvP1 by RFLP analysis was consistent with XX female and XO male genotypes. Analysis of the results of sexual reproduction demonstrated that all progeny inherit the single paternal X chromosome and one of the two maternal X chromosomes. Therefore, all stages of the S. ratti life cycle, with the exception of the free-living males, are XX and genetically female. These findings are considered in relation to previous analyses of S. ratti and to other known sex determination systems.     

Strongyloides spp.: demonstration and partial characterization of acidic collagenolytic activity from infective larvae

Infective larvae of Strongyloides spp. have been shown to contain azocollytic enzymes which may aid in host skin penetration. Attempts to demonstrate classical, neutral pH-active collagenase activity in Strongyloides ratti were unsuccessful. In the current study, we investigated the presence of acidic collagenolytic activity in the infective larvae of Strongyloides ransomi, S. ratti, and S. stercoralis. All three species demonstrated collagenolytic activity in acidic homogenates as well as in neutral freeze-thaw fractions. Biochemical characterization of this collagenolytic activity from S. ratti and S. ransomi indicated that it was active over an acidic pH range, although it was stable at a neutral pH. This, along with molecular weight estimates and inhibitor susceptibilities, suggested that the collagenolytic activity was similar to vertebrate acidic cysteinyl proteinases. These studies also indicated that this activity is similar to the acidic cysteinyl proteinases in extracts of S. ransomi.     

The small subunit ribosomal RNA sequence of Strongyloides stercoralis

The published small subunit rRNA (ssrRNA) gene sequences for Strongyloides ratti and Strongyloides stercoralis are remarkably divergent, particularly in the 5' 400 bases of the approximately 1700 base pair (bp) sequences. This level of divergence between species nominally in the same genus was unprecedented. We have redetermined the ssrRNA sequence of S. stercoralis and find that the published sequence is a chimaera of parasite and fungal segments. The true sequence for S. stercoralis ssrRNA is very similar to that of S. ratti.     

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.     

The control of morph development in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti has a complex life cycle. The progeny of the parasitic females can develop into three distinct morphs, namely directly developing infective third-stage larvae (iL3s), free-living adult males and free-living adult females. We have analysed of the effect of host immune status (an intra-host factor), environmental temperature (an extra-host factor) and their interaction on the proportion of larvae that develop into these three morphs. The results are consistent with the developmental decision of larvae being controlled by at least two discrete developmental switches. One is a sex-determination event that is affected by host immune status and the other is a switch between alternative female morphs that is affected by both host immune status and environmental temperature. These findings clarify the basis of the life cycle of S. ratti and demonstrate how such complex life cycles can result from a combination of simple developmental switches.     

Strongyloides ratti infection in the large intestine of wild rats, Rattus norvegicus

The large intestine of a rat has been neglected almost completely as a site of Strongyloides sp. infection. We reported that adult Strongyloides ratti remained in the large intestine for more than 80 days, producing more number of infective larvae than small intestine adults, and therefore hypothesized that parasitism in this site could be a survival strategy. In wild rats, however, no study has focused on large intestine infections of Strongyloides. The present study revealed that 32.4% of 68 wild rats, Rattus norvegicus, had the infection of S. ratti in the large intestine, with an average of 4.7 worms. These worms harbored normal eggs in the uterus. In a laboratory experiment with S. ratti and Wister rats, daily output of infective larvae by 4.7 females in the large intestine was estimated to be 4,638.4, suggesting that a few parasites could play a role in the parasite transmission. Five species of nematode found in the wild rats showed seasonality in infection intensity, with highest intensities in March-May. The number of S. ratti in the large intestine was also highest in these months.     

The population genetic structure of the facultatively sexual parasitic nematode Strongyloides ratti in wild rats.

We have investigated the population genetic structure of the parasitic nematode Strongyloides ratti in wild rats. In the UK, S. ratti reproduces predominantly by mitotic parthenogenesis, with sexual forms present at a rate of less than 1%. S. ratti was found to be a prevalent parasite and substantial genetic diversity was detected. Most rats were infected with a genotypic mixture of parasites. A hierarchical analysis of the genetic variation found in S. ratti sampled across Britain and Germany showed that 73.3% was explained by variation between parasites within individual hosts and 25.3% by variation between rats within sample sites. Only a small proportion (1.4%) of the total genetic variation was attributable to genetic subdivision between sample sites, suggesting that there is substantial gene flow between these sites. Most parasites sampled were found to exist in Hardy-Weinberg equilibrium and this population genetic structure is discussed in view of the virtual absence of sexual reproduction.     

The beta-tubulin genes of two Strongyloides species

The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the beta-tubulin gene. We have cloned and sequenced the beta-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in beta-tubulin. Our findings indicate that S. ratti has a single beta-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides beta-tubulin gene, thus enabling survey for BZ resistant genotypes.     

Extraordinary plasticity in aging in Strongyloides ratti implies a gene-regulatory mechanism of lifespan evolution

Aging evolves as the result of weakened selection against late-acting deleterious alleles due, for example, to extrinsic mortality. Comparative studies of aging support this evolutionary theory, but details of the genetic mechanisms by which lifespan evolves remain unclear. We have studied aging in an unusual nematode, Strongyloides ratti, to gain insight into the nature of these mechanisms, in this first detailed examination of aging in a parasitic nematode. S. ratti has distinct parasitic and free-living adults, living in the rat small intestine and the soil, respectively. We have observed reproductive and demographic aging in parasitic adults, with a maximum lifespan of 403 days. By contrast the maximum lifespan of free-living adults is only 5 days. Thus, the two adults of S. ratti have evolved strikingly different rates of aging. Parasitic nematode species are frequently longer-lived than free-living species, presumably reflecting different extrinsic mortality rates in their respective niches. Parasitic and free-living female S. ratti are morphologically different, yet genetically identical. Thus, the 80-fold difference in their lifespans, the greatest plasticity in aging yet reported, must largely reflect evolved differences in gene expression. This suggests that interspecific differences in lifespan may evolve via similar mechanisms.     

Strongyloides venezuelensis infections in mice

The course and intensity of Strongyloides venezuelensis infection as compared with S. ratti infection were investigated in BALB/c mice. The mice were found to be much more susceptible to infection with S. venezuelensis than S. ratti. The majority of worms inoculated were recovered from the lungs and subsequently from the small intestines, suggesting that their migratory route via the lungs to the small intestine was comparable to that of S. stercoralis in humans. Spontaneous expulsion of worms occurred by about 10 days after infection, which was the same as that of S. ratti. Different infectivities, as assessed by faecal egg excretion, age, sex and strain of mouse were observed in mice infected with S. venezuelensis, as well as in those infected with S. ratti. A striking immunity was acquired following a primary exposure to S. venezuelensis. Mice infected with S. venezuelensis are considered to provide as useful a model as those infected with S. ratti for the study of human strongyloidiasis.     

Species-specific differences in heterogonic development of serially transferred free-living generations of Strongyloides planiceps and Strongyloides stercoralis

The direction of free-living development (homogonic vs. heterogonic) in Strongyloides stercoralis and Strongyloides planiceps was examined by successive transplantation of the uterine eggs of free-living females into a test tube culture system containing fresh feces. The eggs from the first-generation free-living females of S. stercoralis did not develop into second-generation free-living adult worms, but all developed into filariform larvae. However, the majority of S. planiceps eggs from the first-generation free-living females developed into second-generation free-living adults. By successive transfer of uterine eggs of each generation, 9 generations developed, and in every cycle more adult worms developed than filariform larvae. However, the number of free-living generations was not infinite; in experiments repeated twice, the number of worms developing from the eggs of eighth or ninth generations was too small to continue further culture. These findings indicate that the pattern of free-living development is different between the 2 species.     

Growth of the genital primordium as a marker to describe a time course for the heterogonic larval development in Strongyloides stercoralis

A time course for the heterogonic development of Strongyloides stercoralis is described and a method for distinguishing the early larval stages of this nematode is proposed. The number of cells in the developing gonad were counted at various time intervals of incubation, along with the percentage of larvae in molt at each interval. The time course of growth of the gonad follows a pattern comparable to that reported for body length in an idealized general nematode. A model for the heterogonic development of S. stercoralis is proposed, which, although similar to other nematode developmental models, is stage specific for S. stercoralis, allowing the otherwise morphologically similar rhabditiform stages (L1, L2) to be distinguished.     

Transmammary transmission of Strongyloides stercoralis in dogs

Vertical transmission of larvae is a major pathway in the life cycle of several species of Strongyloides, but evidence for it occurring in humans or dogs with Strongyloides stercoralis is absent. In an effort to determine if vertical transmission could occur with S. stercoralis, each of 3 female dogs was infected with filariform larvae at a different stage of the reproductive cycle, i.e., preconception, gestation, or postpartum. Results showed that none of 6 pups born to a female infected before conception or any of 6 pups born to another female infected during gestation harbored any stage of S. stercoralis when necropsied at parturition. Conversely, all 5 pups that nursed from the female infected immediately postpartum became infected with adult S. stercoralis in their small intestines (range, 56-129 adult worms). Significantly, live filariform larvae of S. stercoralis were observed on 2 different occasions from milk samples taken from the lactating female. Because arrested development of larvae is not known in S. stercoralis, there is no reservoir of larvae in the parenteral tissues of females to queue for passage to the pups and, thus, it is not surprising that only timely infections, perhaps very late in gestation and during lactation, can be successful. These data support previous work in dogs with S. stercoralis, which concluded that vertical transmission through prenatal pathways does not occur, but they are the first from the dog to indicate that vertical transmission of this parasite through transmammary routes is possible. Whether transmammary transmission of S. stercoralis occurs in humans remains unknown but given its immense pathological potential, it should not be overlooked.     

Successful transgenesis of the parasitic nematode Strongyloides stercoralis requires endogenous non-coding control elements

Critical investigations into the cellular and molecular biology of parasitic nematodes have been hindered by a lack of modern molecular genetic techniques for these organisms. One such technique is transgenesis. To our knowledge, the findings reported here demonstrate the first heritable DNA transformation and transgene expression in the intestinal parasite Strongyloides stercoralis. When microinjected into the syncitial gonads of free-living S. stercoralis females, a construct fusing the S. stercoralis era-1 promoter, the coding region for green fluorescent protein (gfp) and the S. stercoralis era-1 3' untranslated region was expressed in intestinal cells of normally developing F1 transgenic larvae. The frequency of transformation and GFP expression among F1 larvae was 5.3%. By contrast, expression of several promoter::gfp fusions incorporating only Caenorhabditis elegans regulatory elements was restricted to abortively developing F1 embryos of S. stercoralis. Despite its lack of regulated expression, PCR revealed that one of these C. elegans-based vector constructs, the sur-5::gfp fusion, is incorporated into F1 larval progeny of microinjected female worms and then transmitted to the F2 through F5 generations during two host passages conducted without selection and punctuated by free-living generations reared in culture. Heritable DNA transformation and regulated transgene expression, as demonstrated here for S. stercoralis, constitute the essential components of a practical system for transgenesis in this parasite. This system has the potential to significantly advance the molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally.     

Use of repeated measure linear modeling to analyze longitudinal data from experimental parasite infections.

Here the use of repeated measure linear modeling to analyze experimental infections is demonstrated. This method of analysis makes full use of serial measurements on the same host throughout the course of an infection while taking into account correlations between measures within the same host. As an example, repeated measure linear modeling is used to analyze worm output from an experimental infection of Strongyloides ratti, a nematode parasite of rats. This analysis allows differences in the temporal dynamics of infection of two lines of S. ratti to be dissected.