Predicting the effects of parasite co-infection across species boundaries.

It is normal for hosts to be co-infected by parasites. Interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. Despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. Here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. From an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. We show that, as predicted, in co-infections, the blood-feeding nematode Haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode Trichostrongylus colubriformis; and that the T. colubriformis-induced immune response negatively affects H. contortus This work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. The study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems.

Copy number variation and genetic diversity of MHC Class IIb alleles in an alien population of Xenopus laevis.

Xenopus laevis (the African clawed frog), which originated through hybridisation and whole genome duplication, has been used as a model for genetics and development for many years, but surprisingly little is known about immune gene variation in natural populations. The purpose of this study was to use an isolated population of X. laevis that was introduced to Wales, UK in the past 50 years to investigate how variation at the MHC compares to that at other loci, following a severe population bottleneck. Among 18 individuals, we found nine alleles based on exon 2 sequences of the Class IIb region (which includes the peptide binding region). Individuals carried from one to three of the loci identified from previous laboratory studies. Genetic variation was an order of magnitude higher at the MHC compared with three single-copy nuclear genes, but all loci showed high levels of heterozygosity and nucleotide diversity and there was not an excess of homozygosity or decrease in diversity over time that would suggest extensive inbreeding in the introduced population. Tajima's D was positive for all loci, which is consistent with a bottleneck. Moreover, comparison with published sequences identified the source of the introduced population as the Western Cape region of South Africa, where most commercial suppliers have obtained their stocks. These factors suggest that despite founding by potentially already inbred individuals, the alien population in Wales has maintained substantial genetic variation at both adaptively important and neutral genes.

The relative contribution of co-infection to focal infection risk in children.

Co-infection is ubiquitous in people in the developing world but little is known regarding the potential for one parasite to act as a risk factor for another. Using generalized linear mixed modelling approaches applied to data from school-aged children from Zanzibar, Tanzania, we determined the strength of association between four focal infections (i.e. Ascaris lumbricoides, Trichuris trichiura, hookworm and self-reported fever, the latter used as a proxy for viral, bacterial or protozoal infections) and the prevalence or intensity of each of the helminth infections. We compared these potential co-infections with additional risk factors, specifically, host sex and age, socioeconomic status and physical environment, and determined what the relative contribution of each risk factor was. We found that the risk of infection with all four focal infections was strongly associated with at least one other infection, and that this was frequently dependent on the intensity of that other infection. In comparison, no other incorporated risk factor was associated with all focal infections. Successful control of infectious diseases requires identification of infection risk factors. This study demonstrates that co-infection is likely to be one of these principal risk factors and should therefore be given greater consideration when designing disease-control strategies. Future work should also incorporate other potential risk factors, including host genetics which were not available in this study and, ideally, assess the risks via experimental manipulation.

Wild mice provide insights into natural killer cell maturation and memory.

The immune system has evolved, and continues to evolve, in response to the selection pressure that infections exert on animals in their natural environments, yet much of our understanding about how the immune system functions comes from studies of model species maintained in the almost complete absence of such environmental selection. The scientific discipline of immunology has among its aims the improvement of human and animal health by the application of immunological knowledge. As research on humans and domesticated animals is highly constrained-ethically, logistically and financially-experimental animal models have become an invaluable tool for dissecting the functioning of the immune system. The house mouse (Mus musculus) is by far the most widely used animal model in immunological research but laboratory-reared mice provide a very narrow view of the immune system-that of a well-fed and comfortably housed animal with minimal exposure to microbial pathogens. Indeed, so much of our immunological knowledge comes from studies of a very few highly inbred mouse strains that-to all intents and purposes-our immunological knowledge is based on enormously detailed studies of very small numbers of individual mice. The limitations of studies in inbred strains of laboratory mice are well-recognized (Pedersen & Babayan 2011), but serious attempts to address these limitations have been few and far between. However, the emerging field of 'ecological immunology' where free-living populations are studied in their natural habitat is beginning to redress this imbalance (Viney et al. 2005; Martin et al. 2006; Owen et al. 2010; Abolins et al. 2011). As demonstrated in the work by Boysen et al. (2011) in this issue of Molecular Ecology, studies in wild animal populations-especially free-living M. musculus-represent a valuable bridge between studies in humans and livestock and studies of captive animals.

Measures of immune function of wild mice, Mus musculus.

The immune function of wild animals has been rather little studied. Wild animals' immune function may differ from that of laboratory bred animals because of their different environments. This idea follows from the concept of resource partitioning in which animals distribute scarce resources to all aspects of life, including to costly immune responses. A logical extension of this idea is that there may be substantial interindividual variation in the immune function of wild animals. To begin to investigate this, we compared the immune function of a laboratory bred mouse strain (C57BL/6, a widely used mouse strain that makes potent immune responses) and wild caught Mus musculus. We found that by most measures of immune function, the wild caught mice had greater immune function. Specifically, wild mice had greater concentrations and more avid antigen-specific IgG responses, as well as higher concentrations of total IgG and IgE, compared with those laboratory bred mice. Moreover, flow cytometric analysis showed a comparatively greater overall level of activation of the cells of the immune system in wild mice. Lastly, we observed that immune function was substantially more variable among wild caught mice than among the laboratory bred mice. The next research challenge is to understand which aspects of an individual animal's life determine its immune function.

How did parasitic worms evolve?

Nematodes are important parasites of humans and other animals. Nematode parasitism is thought to have evolved by free-living, facultatively developing, arrested larvae becoming associated with animals, ultimately becoming parasites. The formation of free-living arrested larvae of the nematode Caenorhabditis elegans is controlled by the environment, and involves dafachronic acid (DA) and transforming growth factor (TGF)-beta signalling. Recent data have shown that DA acid signalling plays a conserved role in controlling larval development in both free-living and parasitic species. In contrast, TGF-beta signalling does not seem to be conserved; this difference perhaps points to how nematode parasitism did evolve.

Detecting interspecific macroparasite interactions from ecological data: patterns and process.

There is great interest in the occurrence and consequences of interspecific interactions among co-infecting parasites. However, the extent to which interactions occur is unknown, because there are no validated methods for their detection. We developed a model that generated abundance data for two interacting macroparasite (e.g., helminth) species, and challenged the data with various approaches to determine whether they could detect the underlying interactions. Current approaches performed poorly - either suggesting there was no interaction when, in reality, there was a strong interaction occurring, or inferring the presence of an interaction when there was none. We suggest the novel application of a generalized linear mixed modelling (GLMM)-based approach, which we show to be more reliable than current approaches, even when infection rates of both parasites are correlated (e.g., via a shared transmission route). We suggest that the lack of clarity regarding the presence or absence of interactions in natural systems may be largely attributed to the unreliable nature of existing methods for detecting them. However, application of the GLMM approach may provide a more robust method of detection for these potentially important interspecific interactions from ecological data.

Natural variation in gene expression in the early development of dauer larvae of Caenorhabditis elegans.

BACKGROUND: The free-living nematode Caenorhabditis elegans makes a developmental decision based on environmental conditions: larvae either arrest as dauer larva, or continue development into reproductive adults. There is natural variation among C. elegans lines in the sensitivity of this decision to environmental conditions; that is, there is variation in the phenotypic plasticity of dauer larva development. We hypothesised that these differences may be transcriptionally controlled in early stage larvae. We investigated this by microarray analysis of different C. elegans lines under different environmental conditions, specifically the presence and absence of dauer larva-inducing pheromone. RESULTS: There were substantial transcriptional differences between four C. elegans lines under the same environmental conditions. The expression of approximately 2,000 genes differed between genetically different lines, with each line showing a largely line-specific transcriptional profile. The expression of genes that are markers of larval moulting suggested that the lines may be developing at different rates. The expression of a total of 89 genes was putatively affected by dauer larva or non-dauer larva-inducing conditions. Among the upstream regions of these genes there was an over-representation of DAF-16-binding motifs. CONCLUSION: Under the same environmental conditions genetically different lines of C. elegans had substantial transcriptional differences. This variation may be due to differences in the developmental rates of the lines. Different environmental conditions had a rather smaller effect on transcription. The preponderance of DAF-16-binding motifs upstream of these genes was consistent with these genes playing a key role in the decision between development into dauer or into non-dauer larvae. There was little overlap between the genes whose expression was affected by environmental conditions and previously identified loci involved in the plasticity of dauer larva development.

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.

Quantitative genetic analysis of life-history traits of Caenorhabditis elegans in stressful environments.

BACKGROUND: Organisms live in environments that vary. For life-history traits that vary across environments, fitness will be maximised when the phenotype is appropriately matched to the environmental conditions. For the free-living nematode Caenorhabditis elegans, we have investigated how two major life-history traits, (i) the development of environmentally resistant dauer larvae and (ii) reproduction, respond to environmental stress (high population density and low food availability), and how these traits vary between lines and the genetic basis of this variation. RESULTS: We found that lines of C. elegans vary in their phenotypic plasticity of dauer larva development, i.e. there is variation in the likelihood of developing into a dauer larva for the same environmental change. There was also variation in how lifetime fecundity and the rate of reproduction changed under conditions of environmental stress. These traits were related, such that lines that are highly plastic for dauer larva development also maintain a high population growth rate when stressed. We identified quantitative trait loci (QTL) on two chromosomes that control the dauer larva development and population size phenotypes. The QTLs affecting the dauer larva development and population size phenotypes on chromosome II are closely linked, but are genetically separable. This chromosome II QTL controlling dauer larva development does not encompass any loci previously identified to control dauer larva development. This chromosome II region contains many predicted 7-transmembrane receptors. Such proteins are often involved in information transduction, which is clearly relevant to the control of dauer larva development. CONCLUSION: C. elegans alters both its larval development and adult reproductive strategy in response to environmental stress. Together the phenotypic and genotypic data suggest that these two major life-history traits are co-ordinated responses to environmental stress and that they are, at least in part, controlled by the same genomic regions.

Genes important in the parasitic life of the nematode Strongyloides ratti.

Parasitic nematodes are important pathogens of humans and other animals. The genus Strongyloides has both a parasitic and a free-living adult generation. S. ratti infections of its rat host are negatively affected by the host immune response, such that a month after infection, worms are lost from the hosts. Here we have investigated the changes in parasite gene expression that occur as the anti-S. ratti immune pressure increases. Existing S. ratti expressed sequence tags were used to construct a microarray consisting of 2227 putative genes. This was probed with cDNA prepared from parasites subject to low or high immune pressures. There are significant changes in the gene expression of S. ratti when subject to different immune pressures. Most of the genes whose expression changes have no significant alignment to known genes. These data together with previous S. ratti EST data were then used to identify genes that we hypothesise are central to the parasitic life of S. ratti and, perhaps, other parasitic nematodes. These analyses have identified genes likely to play a key role in the parasitic life of S. ratti; these genes should be the priority for further investigation.

Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti.

The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown rats. Specifically, gender-biased transcription between free-living females and free-living males, and parasitic-biased transcription between parasitic females and free-living females was determined. Of the estimated 3,688 distinct transcripts represented on the microarray, 743 (20%) exhibited male-biased transcription of >1.4-fold (2(0.5)), 689 (19%) female-biased transcription, 418 (11%) parasitic-biased transcription and 305 (8%) free-living-biased transcription. Among those transcripts that exhibited the highest levels of differential transcription, an orthologue of major sperm protein was identified in males, distinct aspartic protease orthologues in either parasitic or in free-living females, and orthologues of hsp-17 chaperone in parasitic females. These 3,688 transcripts were separated into 12 clusters, such that the pattern of transcription between life-stages and biological replicates was similar among transcripts within a cluster and dissimilar between clusters. Using annotation inferred from Caenorhabditis elegans, gene ontology terms over-represented in one or more clusters were identified and showed that female-biased transcription was associated with genes involved in reproductive processes and larval development, male-biased transcription was linked to genes involved in metabolism, and free-living-biased transcription related to genes involved in the regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased transcription was less clear. The present findings for S. ratti provide a basis for a detailed exploration of differentially regulated molecules and might assist in the search for novel drug or vaccine targets in parasitic nematodes.

Density-dependent immune responses against the gastrointestinal nematode Strongyloides ratti.

Negative density-dependent effects on the fitness of parasite populations are an important force in their population dynamics. For the parasitic nematode Strongyloides ratti, density-dependent fitness effects require the rat host immune response. By analysis of both measurements of components of parasite fitness and of the host immune response to different doses of S. ratti infection, we have identified specific parts of the host immune response underlying the negative density-dependent effects on the fitness of S. ratti. The host immune response changes both qualitatively from an inflammatory Th1- to a Th2-type immune profile and the Th2-type response increases quantitatively, as the density of S. ratti infection increases. Parasite survivorship was significantly negatively related to the concentration of parasite-specific IgG(1) and IgA, whereas parasite fecundity was significantly negatively related to the concentration of IgA only.

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.

The comparative immunology of wild and laboratory mice, Mus musculus domesticus.

The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.

A microarray analysis of gene expression in the free-living stages of the parasitic nematode Strongyloides ratti.

BACKGROUND: The nematode Strongyloides ratti has two adult phases in its lifecycle: one obligate, female and parasitic and one facultative, dioecious and free-living. The molecular control of the development of this free-living generation remains to be elucidated. RESULTS: We have constructed an S. ratti cDNA microarray and used it to interrogate changes in gene expression during the free-living phase of the S. ratti life-cycle. We have found very extensive differences in gene expression between first-stage larvae (L1) passed in faeces and infective L3s preparing to infect hosts. In L1 stages there was comparatively greater expression of genes involved in growth. We have also compared gene expression in L2 stages destined to develop directly into infective L3s with those destined to develop indirectly into free-living adults. This revealed relatively small differences in gene expression. We find little evidence for the conservation of transcription profiles between S. ratti and S. stercoralis or C. elegans. CONCLUSION: This is the first multi-gene study of gene expression in S. ratti. This has shown that robust data can be generated, with consistent measures of expression within computationally determined clusters and contigs. We find inconsistencies between EST representation data and microarray hybridization data in the identification of genes with stage-specific expression and highly expressed genes. Many of the genes whose expression is significantly different between L1 and iL3s stages are unknown beyond alignments to predicted genes. This highlights the forthcoming challenge in actually determining the role of these genes in the life of S. ratti.

daf-7 and the development of Strongyloides ratti and Parastrongyloides trichosuri.

daf-7 is a key ligand in one of the three pathways that control dauer larva development in Caenorhabditis elegans. Given the similarities between dauer larvae of free-living nematodes and third stage infective larvae of animal parasitic nematodes, we hypothesised that daf-7 may be involved in the development of these infective larvae. To investigate this, we cloned daf-7 orthologues from Strongyloides ratti and Parastrongyloides trichosuri and analysed their RNA level by semi-quantitative RT-PCR during the S. ratti and P. trichosuri life cycles and in a range of in vitro and in vivo conditions. We found that, in both species, the RNA level of daf-7 was low in free-living stages but peaked in the infective L3 (iL3) stage with little or no expression in the parasitic stages. This contrasts with the daf-7 RNA level in C. elegans, which peaks in L1, decreases thereafter, and is absent in dauer larvae. The RNA level of daf-7 in infective larvae was reduced by larval penetration of host skin or development in the host, but not by a shift to the body temperature of the host.

An expressed sequence tag analysis of the life-cycle of the parasitic nematode Strongyloides ratti.

14,761 expressed sequence tags (ESTs) were generated, representing five stages during the parasitic and free-living phases of the life-cycle of the parasitic nematode Strongyloides ratti. These ESTs formed 4152 clusters, of which 97% contained 10 or fewer ESTs and 66% were singletons. These 4152 clusters are likely to represent approximately 20% of S. ratti's genes. The clusters' consensus sequences were used to assign each cluster to one of three databases: (i) Caenorhabditis elegans and C. briggsae sequences; (ii) other nematode sequences; (iii) non-nematode sequences. This approach has identified putative nematode-specific genes, that may be targets for developing approaches for parasitic nematode control. Approximately 25% of the clusters have no significant alignments and may therefore represent novel genes. The EST representation between the libraries was used to analyse stage-specific or -biased expression in silico. This showed that 81% of clusters are present in only one library and 12% are present in any two libraries, indicating substantial stage-specificity of gene expression. The 30-most abundantly expressed clusters were analysed in further detail. Many of these have significantly different parasitic- or free-living-specific or -biased expression. Many of the parasitic-specific genes are, as yet, uncharacterised: one of these represents 25% of all ESTs obtained from the parasitic stage.

The biology of Strongyloides spp.

Strongyloides is a genus of parasitic nematodes that, unusually, has a free-living adult generation. Here we introduce the biology of this genus, especially the fascinating but complex life-cycle, together with an overview of the taxonomy, morphology, genetics, and genomics of this genus.

Extinction of an introduced warm-climate alien species, Xenopus laevis, by extreme weather events.

Invasive, non-native species represent a major threat to biodiversity worldwide. The African amphibian Xenopus laevis is widely regarded as an invasive species and a threat to local faunas. Populations originating at the Western Cape, South Africa, have been introduced on four continents, mostly in areas with a similar Mediterranean climate. Some introduced populations are also established in cooler environments where persistence for many decades suggests a capacity for long-term adaptation. In these cases, recent climate warming might enhance invasion ability, favouring range expansion, population growth and negative effects on native faunas. In the cool temperate UK, populations have been established for about 50 years in Wales and for an unknown period, probably >20 years, in England (Lincolnshire). Our field studies over 30 and 10 years, respectively, show that in favourable conditions there may be good recruitment, fast individual growth rates and large body size; maximum longevity exceeds 23 years. Nevertheless, areas of distribution remained limited, with numbers <500 in each population. In 2010, only a single individual was captured at each locality and further searching failed to record any others in repeated sampling up to 2014. We conclude that both populations are now extinct. The winters of 2009-2010 and 2010-2011 experienced extreme cold and drought (December 2010 was the coldest in 120 years and the third driest in 100 years). The extinction of X. laevis in these areas indicates that even relatively long-established alien species remain vulnerable to rare extreme weather conditions.