Male Parasites Research Articles

An effective sequence characterized amplified region‐PCR method derived from restriction site‐amplified polymorphism for the identification of femaleSchistosoma japonicumof zoonotic significance

In the present study, restriction site-amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8+R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re-amplified and sequenced, and a sequence of 162 bp was obtained. Based on this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)-PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR-PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR-PCR assay was 0.3 ng for female S. japonicum. The SCAR-PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR-PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.

Heterogeneity of class I and class II MHC sequences in Schistosoma japonicum from different endemic regions in mainland China

The present study examined sequence variation in class I and class II major histocompatibility complex (MHC) genes among Schistosoma japonicum isolates from different endemic regions in mainland China and assessed the level of horizontal gene transfer and sequence similarity between parasites and their hosts. S. japonicum cercariae were used to infect male adult rabbits to obtain adult S. japonicum samples. A portion of the class I MHC gene (pMHC I) and class II MHC genes (pMHC II) were amplified separately from individual adult trematodes by polymerase chain reaction and sequenced. Among all the examined isolates of S. japonicum, sequence differences between male and female parasites were 0.0-26.6% for pMHC I and 0.0-7.0% for pMHC II. Sequence variations between male and female parasites among different geographical locations from the mountainous areas were 1.1-26.6% for pMHC I and 1.5-3.0% for pMHC II. Sequence variations between samples from Yunnan and those from Sichuan were 2.7-23.5% for pMHC I and 1.1-3.7% for pMHC II. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-25.0% for pMHC I and 0.0-7.0% for pMHC II. Sequence variations between S. japonicum isolates from mountainous areas, and those from lake/marshland areas were 0.0-26.1% for pMHC I and 0.4-6.1% for pMHC II. BLASTN analysis indicated that all the pMHC II sequences showed high homology to a portion of exon 3 in rabbit MHC class II DP beta gene with more than 89% similarity, and all the pMHC I sequences except isolates in Yunnan (Eryuan) revealed high homology to the portion of exon 2 in rabbit MHC I gene with more than 81% similarity. Phylogenetic analysis showed no specific clustering comprising parasites from single geographical or endemic regions, and the paired parasites were even found in different clusters. These results demonstrated that pMHC I and II of S. japonicum isolates in mainland China existed heterogeneity, but the pMHC I, II, or combined sequences were not suitable markers for examining genetic relationship among different isolates from endemic regions in mainland China.

Geographic variation in sterilizing parasite species and the Red Queen

The Red Queen hypothesis predicts that sexual reproduction should be favoured in locations where the risk of infection by virulent parasites is consistently high. When hosts are exposed to multiple parasites over their geographic range, the coevolving parasite species may vary among host populations. We surveyed 26 streams on the South Island of New Zealand to determine whether the frequency of snails (Potamopyrgus antipodarum) infected by various sterilizing trematode parasite species was correlated with the frequency of sexual individuals. We compared the results with a survey conducted over 20 years ago to determine whether the associations were consistent. We also evaluated different measures of parasite‐mediated selection among populations, including prevalence of the most common local parasite (MCLP) species and parasite diversity to assess the best predictor of sexual reproduction among stream populations. The results showed that the relationship between male frequency and parasite infection is more geographically widespread than previously recorded. Additionally, we found that the prevalence of the MCLP was the best predictor of sex in habitats where hosts populations are infected with multiple parasites (approximately 15 trematode species). This study provides evidence that sexual snails occur more often in environments with high infection levels, and that the pattern of parasite‐imposed selection is geographically variable. Support for the Red Queen may be strengthened by focussing on the MCLP, which may vary among host populations.

Changes in Gene Expression in European Eels (Anguilla anguilla) Induced by Infection with Swim Bladder Nematodes (Anguillicola crassus)

The European eel, Anguilla anguilla, is considered an endangered species that is under pressure for many reasons. Among others, the introduced parasite Anguillicola crassus is thought to play an important role in the decline of eel populations. These nematodes have been shown to negatively affect many fitness-related traits in eels, e.g., growth, osmoregulation, and stress tolerance. Nevertheless, there has been little work on the way in which the host-parasite interaction influences the molecular regulation of these key physiological processes. We experimentally analyzed the effect of this nematode on the expression of genes involved in the physiology of European eels during their continental life. Included are genes that are implicated in the eel's somatic growth (insulin-like growth factor 1 and thyroid hormone receptor beta), osmoregulation (Na+/K+-ATPase beta1 and aquaporin 3), and hematopoiesis (hemoglobin alpha-chain). Our results showed the absence of an effect on genes involved in fish growth; the parasite may, however, have an effect on osmoregulation and hematopoiesis. We also noted a differential impact of male and female parasites on the expression of some genes, perhaps owing to the sexual dimorphism in body size of the parasite.

Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development

Microarray analysis was used to investigate differential gene regulation in adult male and female Schistosoma japonicum cultured in the presence or absence of insulin in vitro. A total of 1,101 genes were up- or down-regulated in response to insulin, the majority of differential expression occurring 24 h after the addition of insulin to the cultures. Genes differentially expressed in male or female worms were predominantly involved in growth and development, with significant sex-specific differences in transcriptional profiles evident. Insulin appeared to promote protein synthesis and control protein degradation more prominently in male parasites. The study also indicated that insulin plays a more pronounced role in the uptake of glucose in unpaired female parasites, as reflected in the increased stimulation of gene expression of the phosphatidylinositol 3-kinase sub-pathway of insulin signalling. Insulin may also impact on the sexual differentiation and fecundity of female schistosomes by activation of the mitogenic-activated protein kinase sub-pathway.

A diagnostically useful 200-kDa protein is secreted through the surface pores of the filarial parasite Setaria digitata

The excretory/secretory (ES) materials from filarial parasites form an important tool for immunodiagnosis of filariasis. We have raised monoclonal antibodies against ES proteins isolated from the medium incubated with live adult bovine filarial parasite Setaria digitata. The hybridoma were cloned and characterised with respect to the individual proteins of the ES materials. A secretory glycoprotein with molecular weight 200-kDa (gp200) was purified, localised and characterised using the specific monoclonal antibodies raised against it. The immunolocalisation study clearly showed that the protein is secreted out through the pores on the surface of both male and female parasites. The gp200 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed dissociation into 11- to 66-kDa 'ladder' polypeptides, and all of them could be identified with the same monoclonal antibody. The gp200 is normally shed along with the ES materials. The screening of human sera samples using the monoclonal antibodies give promising results which showed that gp200 could be a potent candidate protein for the immunodiagnosis of filariasis by the method of antigen detection.

Acanthocephalan size and sex affect the modification of intermediate host colouration

For trophically transmitted parasites, transitional larval size is often related to fitness. Larger parasites may have higher establishment success and/or adult fecundity, but prolonged growth in the intermediate host increases the risk of failed transmission via natural host mortality. We investigated the relationship between the larval size of an acanthocephalan (Acanthocephalus lucii) and a trait presumably related to transmission, i.e. altered colouration in the isopod intermediate host. In natural collections, big isopods harboured larger worms and had more modified (darker) abdominal colouration than small hosts. Small isopods infected with a male parasite tended to have darker abdominal pigmentation than those infected with a female, but this difference was absent in larger hosts. Female size increases rapidly with host size, so females may have more to gain than males by remaining in and growing mutually with small hosts. In experimental infections, a large total parasite volume was associated with darker host respiratory operculae, especially when it was distributed among fewer worms. Our results suggest that host pigment alteration increases with parasite size, albeit differently for male and female worms. This may be an adaptive strategy if, as parasites grow, the potential for additional growth decreases and the likelihood of host mortality increases.

The Impact of Environmental Heterogeneity on Genetic Architecture in a Wild Population of Soay Sheep

This work demonstrates that environmental conditions experienced by individuals can shape their development and affect the stability of genetic associations. The implication of this observation is that the environmental response may influence the evolution of traits in the wild. Here, we examined how the genetic architecture of a suite of sexually dimorphic traits changed as a function of environmental conditions in an unmanaged population of Soay sheep (Ovis aries) on the island of Hirta, St. Kilda, northwest Scotland. We examined the stability of phenotypic, genetic, and environmental (residual) covariance in males during the first year of life between horn length, body weight, and parasite load in environments of different quality. We then examined the same covariance structures across environments within and between the adult sexes. We found significant genotype-by-environment interactions for lamb male body weight and parasite load, leading to a change in the genetic correlation among environments. Horn length was genetically correlated with body weight in males but not females and the genetic correlation among traits within and between the sexes was dependent upon the environmental conditions experienced during adulthood. Genetic correlations were smaller in more favorable environmental conditions, suggesting that in good environments, loci are expressed that have sex-specific effects. The reduction in genetic correlation between the sexes may allow independent evolutionary trajectories for each sex. This study demonstrates that the genetic architecture of traits is not stable under temporally varying environments and highlights the fact that evolutionary processes may depend largely upon ecological conditions.

Effects of low-protein diet onSchistosoma mansonimorphology visualized by morphometry and confocal laser scanning microscopy

Previous studies have shown that protein deficiencies can hamper both the course of experimental schistosomiasis and normal development of adult worms. To further investigate this relationship, we compared adult male and female Schistosoma mansoni from malnourished and well-fed mice through morphometric and confocal laser scanning microscopy analysis. Swiss mice were fed protein-deficient diets (8%) and infected subcutaneously with approximately 80 S. mansoni cercariae (BH strain, Brazil). Control mice were fed a standard rodent diet (23% protein). The nutritional status was evaluated by body weight gain and albumin values. Mice were sacrificed 63 days post-infection. Recovered worms were stained with hydrochloric carmine and preserved as whole-mounts for bright-field examination and confocal microscopy. The body weight gain and serum albumin concentrations were significantly lower (P < 0.05) in malnourished mice than in controls. In general, all morphometric values of specimens grown in malnourished mice were lower than those of control mice. Schistosome worms grown in malnourished mice had statistically significant differences (P < 0.05) in the reproductive system and tegument than those grown in mice fed standard diets. In female worms, vitelline glands showed few remaining follicles and ovaries lacked mature oocytes. In male parasites, tubercles were fewer in number on the dorsal surface and testicular lobes presented fewer differentiated germinal cells. In summary, we describe novel data supporting the view that low-protein diets may influence the development of adult worms.

Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 ( cox3), NADH dehydrogenase subunits 4 and 5 ( nad4 and nad5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox3 gene (p cox3), a portion of the nad4 and nad5 genes (p nad4 and p nad5) were amplified separately from individual trematodes by polymerase chain reaction (PCR) and the amplicons were subjected to direct sequencing. In the mountainous areas, sequence variations between parasites from Yunnan and those from Sichuan were 0.3% for p cox3, 0.0–0.1% for p nad4, and 0.0–0.2% for p nad5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0–0.3% for p cox3, 0.0–0.7% for p nad4, and 0.0–1.6% for p nad5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0–0.5% for p cox3, 0.0–0.7% for p nad4, and 0.0–1.6% for p nad5. Phylogenetic analyses based on the combined sequences of p cox3, p nad4 and p nad5 revealed that S. japonicum isolates from mountainous areas (Yunnan and Sichuan provinces) clustered together. For isolates from the lake/marshland areas, isolates from Anhui and Jiangsu provinces clustered together and was sister to samples from Jiangxi province, while isolates from Hubei and Zhejiang province clustered together. However, isolates from different geographical locations in Hunan province were in different clades. These findings demonstrated the usefulness and attributes of the three mtDNA sequences for population genetic studies of S. japonicum, and have implications for studying population biology, molecular epidemiology, and genetic structure of S. japonicum, as well as for the effective control of schistosomiasis.

PTK‐pathways and TGF‐β signaling pathways in schistosomes

Schistosome parasites have co-evolved an intricate relationship with their human and snail hosts as well as a novel interplay between the adult male and female parasites. Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. Studies on signaling in schistosomes opens a new era for investigation of host-parasite and male-female interactions. We review the role of the TGF-beta signaling pathway in parasite development, host-parasite interactions and male-female interactions. This review also summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, cytoplasmatic tyrosine kinases of the Src class seem to play especially dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGF-beta signaling in the production of vitellocytes. Finally, we take advantage of the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.

Long-term effects of early parasite exposure on song duration and singing strategy in great tits

Song is a sexually selected trait in many bird species and has been suggested to function as a signal of a male's health and parasite resistance. Here we present an experimental field study on the long-term effects of parasite exposure early in life on adult bird song. We exposed nestling great tits (Parus major) to ectoparasitic hen fleas (Ceratophyllus gallinae) and on their recruitment into the local breeding population assessed the response to a playback of a challenging male in their breeding territory. We show, to our knowledge for the first time in a wild bird population, that parasite exposure early in life affects bird song: song duration of males that were exposed to parasites early in life was reduced by 32% compared with males that grew up in a flea-free environment. Early parasite exposure also significantly reduced the degree of song overlap with the playback, which has been shown to correlate with social status. There was no effect of early parasite exposure on the number of different song types sung or on the latency until the males started the vocal response to the playback. These results suggest that mates or rivals can use song duration and song overlap as proxies for an individual's exposure to parasites early in life. It thereby highlights both the importance of parasites in maintaining honesty of sexually selected traits and the costs of parasitism in terms of reduced attractiveness and competitiveness.

Transmission risk of human trichinellosis

Trichinella is a food-borne parasitic zoonoses and human cases are still reported in Europe mainly due to the consumption of pig meat originating from small backyard farms. Infections originating from industrialized pig farming have not been reported for decades in Europe, due to control measures to prevent the transmission of Trichinella from wildlife by indoor housing and good management practices. Therefore, risk-based monitoring programs might replace individual carcass control in industrialized pig farming as described in EU legislation SANCO 2075/2005. Transmission of Trichinella species between wildlife and the risk that may pose to humans via consumption of contaminated pork meat has not been studied quantitatively. One pathway by which human trichinellosis can occur is the rat–pig–human route. To evaluate the transmission risk though this pathway the dose responses of rat, pig, and human were studied. Experimental T. spiralis infection was performed in rats with doses of as few as 10 parasites and the data set was analysed using a newly developed dose response model that describes larvae per gram (LPG). Experimental T. spiralis infection in pig was analysed in a similar way. Furthermore nine published outbreaks of human trichinellosis were analysed to determine the dose response in humans. The risk of human trichinellosis via the rat–pig–human transmission was simulated by the Monte Carlo method. A pair of female and male parasites representing the lowest infection pressure from the environment, led to the probability of human trichinellosis by consumption of 100g of raw pork meat equal to 5% via the studied rat–pig–human pathway. In the absence of rodent control near the farm, a low infection pressure from wildlife presents a relatively high risk of human trichinellosis via consumption of uncooked pork meat.

Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins

Major sperm proteins (MSPs) represent a protein family occurring in nematodes only. Identification of the 3' and 5' untranslated region (UTR) completed the so far partial msp complementary DNA sequences of the bovine lungworm Dictyocaulus viviparus. The full-length transcript contains sequence tracts consistent with the Kozak and polyadenylation consensus sequence. On genomic level, three full-length sequences differing in three nucleotides were determined containing a 65-bp phase zero intron. Conceptual translation inferred two MSP isoforms due to one substitution within the 126-amino acid polypeptide. Bioinformatic analysis predicted that bovine lungworm MSP folds into an immunoglobulin-like seven-stranded beta sandwich as known for Caenorhabditis elegans and Ascaris suum. Furthermore, bovine lungworm MSP is confidentially predicted to be N-terminal-acetylated and secreted via a non-classical pathway. Quantitative real-time polymerase chain reaction analysis using ten developmental lungworm stages showed that msp is transcribed mainly in adult male parasites and in some degree in hypobiotic L5. However, marginal msp transcription was detectable in all of the investigated developmental lungworm stages.

Schistosoma mansoni: Microarray analysis of gene expression induced by host sex

Schistosoma mansoni is a digenetic trematode and a human parasite responsible for high social and economic impact. Although some authors have studied the effect of host hormones on parasites, not much is known about the effects of host sex on gene expression in Schistosomes. In order to study gene transcripts associated with the host sex, we compared the gene expression profiles of both male and female unisexual adult S. mansoni parasites raised on either male or female hosts, using DNA microarrays. Our results show that host sex caused differential expression of at least 11 genes in female parasites and of 134 in male parasites. Of the differentially expressed genes in female worms, 10 were preferentially expressed in female worms from male mice, while of the 134 differentially expressed genes in male parasites, 79 (59%) were preferentially expressed in worms from female mice. Further investigation of the role of each of those genes will help understand better their importance in the pathogenesis of Schistosomiasis.

A bone morphogenetic protein homologue in the parasitic flatworm, Schistosoma mansoni

Members of the bone morphogenetic protein (BMP) subfamily of cytokines control many aspects of metazoan development including patterning and organogenesis. Despite the recognition that schistosomes possess key components of a BMP signaling pathway, a BMP-like ligand in the parasitic flatworm Schistosoma mansoni remained elusive. Here, we describe the cloning and characterisation of an S. mansoni BMP ( SmBMP). SmBMP is most closely related to BMP homologues from the free-living flatworms Schmidtea mediterranea and Dugesia japonica, with 51% and 47% identity at the amino acid level, respectively. Based on reverse transcription-PCR, SmBMP is expressed throughout the mammalian life-cycle of the parasite in both male and female schistosomes. In support of these results, antibodies to SmBMP successfully immunoprecipitated the protein in adult male and female antigen preparations with more protein detected in male parasites. Immunofluorescent studies localised SmBMP to the protonephridia of adult parasites, and SmBMP was identified in the excretory/secretory products of adult male parasites via immunoprecipitation. With the previous description of a TGF-β subfamily homologue in S. mansoni, ligands representing both arms of the TGF-β superfamily have now been described in this trematode.

Do Hosts Discriminate between Sexually Dichromatic Male and Female Brown‐headed Cowbirds?

AbstractFemale brood parasites are recognized as threats to reproductive success by many host species. Male brood parasites may accompany females while they search for nests to parasitize and males depredate nests throughout the nesting cycle. Hence, selection may also favour recognition of males. We examined whether two common host species perceive male brown‐headed cowbirds (Molothrus ater) as brood parasites, as nest predators, or neither. We quantified visits of male cowbirds to nests of yellow warblers (Dendroica petechia) and red‐winged blackbirds (Ageliaus phoeniceus) to assess the frequency with which these host species interact with male cowbirds. Males were observed near nests during hosts’ laying and incubating stages, although less frequently than female cowbirds. No visits by cowbirds occurred while parents cared for nestlings. We then presented models of male and female cowbirds plus a non‐threatening control to yellow warblers and red‐winged blackbirds during laying and nestling periods. If hosts perceive males and females similarly, they should respond more intensely to the cowbird models during the laying period, when nests are most likely to be parasitized. Both species responded similarly to male and female cowbird models during laying, which suggests that hosts view cowbirds of both sexes as threats. The responses of yellow warblers with nestlings to male cowbirds were strongly influenced by the order of model presentation. Warblers first presented with the male cowbird gave much reduced anti‐parasite responses than those that first interacted with the female then the male cowbird. These results suggest that yellow warblers recognized male vs. female cowbirds, but that discrimination was not expressed during laying. By contrast, red‐winged blackbirds did not discriminate between male and female cowbirds at either nesting stage.

Horns honestly advertise parasite infection in male and female African buffalo

The evolution and maintenance of elaborate secondary sexual characters in males have been the subject of intense interest since Darwin's time. Parasite-mediated sexual selection (PMSS) suggests that elaborate ornaments serve as honest indicators of male health and parasite resistance. Studies testing this key prediction of PMSS have largely focused on ornaments, with the role parasites might play in the maintenance of elaborate weapons being relatively understudied. Here, we tested whether weapon (horn) size was an indicator of health status in male and female African buffalo, Syncerus caffer. We examined whether individuals with larger horns were less likely to be infected with parasites, and had lower parasite loads and stronger immune systems. In males, horn size was significantly negatively correlated with the number of different parasites infecting an individual (parasite richness), the likelihood of infection with strongyle nematodes and coccidia, and strongyle intensity. In females, horn size was significantly negatively correlated with parasite richness, occurrence of coccidia infection, coccidia intensity and white blood cell count. These findings were robust when the effects of body condition, age and season were controlled for, consistent with the idea that horns function as honest indicators of health in both sexes. Our study provides new insight into the evolution and maintenance of elaborate weapons in mammals, suggesting a role for PMSS in both males and females.

Phenotypic correlates and survival consequences of male mating success in lek-mating greater prairie-chickens (Tympanuchus cupido)

Female choice and male–male aggression are two modes of sexual selection that can lead to elaboration of male morphological and behavioral traits. In lek-mating species, male mating success is often strongly skewed, and it is puzzling why variation in male traits is still observed given directional female choice. If male traits correlated with reproductive success are honest signals of male quality, there may be survival costs associated with the expression of those traits. In this study, we examined whether morphological, behavioral, and territorial traits are correlated with male mating success and survival in the lek-mating greater prairie-chicken (Tympanuchus cupido). We introduce a novel application of multinomial discrete choice models for analysis of female mate choice behavior. We found that behavioral and territorial attributes showed 6.5 times more variability among males than morphological traits. Both display and aggressive behaviors were strong predictors of male mating success, suggesting that both female choice and male–male aggression were important in determining mating success among male greater prairie-chickens. Moreover, annual survival of male prairie-chickens was independent of mating success and male traits. Females appear to be choosing males based on behavioral traits where large variation exists between males (coefficient of variation >30%). Behavioral traits were the most important factor in determining mating success of male prairie-chickens, but the mechanism underlying this relationship is unknown. In the future, experimental manipulations of male hormones or parasite loads could bridge the proximate mechanisms and ultimate consequences of factors mediating male mating success in lek-mating grouse.

Sympatric speciation

Sympatric speciation