Host phenotype and microbiome vary with infection status, parasite genotype, and parasite microbiome composition.

A growing literature demonstrates the impact of helminths on their host gut microbiome. We investigated whether the stickleback host microbiome depends on ecoevolutionary variables by testing the impact of exposure to the cestode parasite Schistocephalus solidus with respect to infection success, host genotype, parasite genotype, and parasite microbiome composition. We observed constitutive differences in the microbiome of sticklebacks of different origin, and those differences increased when sticklebacks exposed to the parasite resisted infection. In contrast, the microbiome of successfully infected sticklebacks varied with parasite genotype. More specifically, we revealed that the association between microbiome and immune gene expression increased in infected individuals and varied with parasite genotype. In addition, we showed that S. solidus hosts a complex endomicrobiome and that bacterial abundance in the parasite correlates with expression of host immune genes. Within this comprehensive analysis we demonstrated that (i) parasites contribute to modulating the host microbiome through both successful and unsuccessful infection, (ii) when infection is successful, the host microbiome varies with parasite genotype due to genotype-dependent variation in parasite immunomodulation, and (iii) the parasite-associated microbiome is distinct from its host and impacts the host immune response to infection.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

Development of 14 Microsatellite Markers for Zoonotic Tapeworm Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidea).

Dibothriocephalus dendriticus is one of the causative agents of the fish-borne zoonosis diphyllobothriosis. Polymorphic microsatellite markers were originally developed for future genetic studies using microsatellite library screening and next-generation sequencing (NGS). Out of 128 microsatellite candidates selected after NGS analysis, 126 yielded PCR products of the expected size. A declared repetitive motif was confirmed in 92 loci by Sanger sequencing. The level of polymorphism was tested by fragment analysis. Statistical tests for observed and expected heterozygosities and deviations from Hardy-Weinberg equilibrium revealed 14 polymorphic microsatellite loci suitable for studies on the finer genetic structure of global populations of D. dendriticus.

A novel C-type lectin gene is a strong candidate gene for Benedenia disease resistance in Japanese yellowtail, Seriola quinqueradiata.

Little is known about mechanisms of resistance to parasitic diseases in marine finfish. Benedenia disease is caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail/amberjack Seriola quinqueradiata. To uncover the bioregulatory mechanism of Benedenia disease resistance, complete Illumina sequencing of BAC clones carrying genomic DNA for the QTL region in linkage group Squ2 was performed to reveal a novel C-type lectin in this region. Expression of the mRNA of this C-type lectin was detected in skin tissue parasitized by B. seriolae. Scanning for single nucleotide polymorphisms (SNPs) uncovered a SNP in the C-type lectin/C-type lectin-like domain that was significantly associated with B. seriolae infection levels. These results strongly suggest that the novel C-type lectin gene controls resistance to Benedenia disease in Japanese yellowtails.

Discordant population histories of host and its parasite: A role for ecological permeability of extreme environment?

Biogeographical and ecological barriers strongly affect the course of micro-evolutionary processes in free living organisms. Here we assess the impact of a recently emerged barrier on populations of limnic fauna. Genetic diversity and population structure in a host-parasite system (Wenyonia virilis tapeworm, Synodontis schall catfish) are analyzed in the recently divided Turkana and Nile basins. The two basins, were repeatedly connected during the Holocene wet/dry climatic oscillations, following late Pleistocene dessication of the Turkana basin. Mitochondrial DNA sequences for cytochrome oxidase I gene (cox I) and a whole genome scanning method-amplified fragment length polymorphism (AFLP) were employed. A total of 347 cox I sequences (representing 209 haplotypes) and 716 AFLP fragments, as well as 120 cox I sequences (20 haplotypes) and 532 AFLP fragments were obtained from parasites and hosts, respectively. Although results indicate that host and parasite populations share some formative traits (bottlenecks, Nilotic origin), their population histories/patterns differ markedly. Mitochondrial analysis revealed that parasite populations evolve significantly faster and show remarkably higher genetic variability. Analyses of both markers confirmed that the parasites undergo lineage fission, forming new clusters specific for either freshwater or saline parts of Lake Turkana. In congruence with the geological history, these clusters apparently indicate multiple colonisations of Lake Turkana from the Nile. In contrast, the host population pattern indicates fusion of different colonisation waves. Although fish host populations remain connected, saline habitats in Lake Turkana (absent in the Nile), apparently pose a barrier to the gene flow in the parasite, possibly due to its multihost lifecycle, which involves freshwater annelids. Despite partially corroborating mitochondrial results, AFLP data was not sufficiently informative for analyzing populations with recently mixed biogeographic histories.

Landscape genetics of Schistocephalus solidus parasites in threespine stickleback (Gasterosteus aculeatus) from Alaska.

The nature of gene flow in parasites with complex life cycles is poorly understood, particularly when intermediate and definitive hosts have contrasting movement potential. We examined whether the fine-scale population genetic structure of the diphyllobothriidean cestode Schistocephalus solidus reflects the habits of intermediate threespine stickleback hosts or those of its definitive hosts, semi-aquatic piscivorous birds, to better understand complex host-parasite interactions. Seventeen lakes in the Cook Inlet region of south-central Alaska were sampled, including ten in the Matanuska-Susitna Valley, five on the Kenai Peninsula, and two in the Bristol Bay drainage. We analyzed sequence variation across a 759 bp region of the mitochondrial DNA (mtDNA) cytochrome oxidase I region for 1,026 S. solidus individuals sampled from 2009-2012. We also analyzed allelic variation at 8 microsatellite loci for 1,243 individuals. Analysis of mtDNA haplotype and microsatellite genotype variation recovered evidence of significant population genetic structure within S. solidus. Host, location, and year were factors in structuring observed genetic variation. Pairwise measures revealed significant differentiation among lakes, including a pattern of isolation-by-distance. Bayesian analysis identified three distinct genotypic clusters in the study region, little admixture within hosts and lakes, and a shift in genotype frequencies over time. Evidence of fine-scale population structure in S. solidus indicates that movement of its vagile, definitive avian hosts has less influence on gene flow than expected based solely on movement potential. Observed patterns of genetic variation may reflect genetic drift, behaviors of definitive hosts that constrain dispersal, life history of intermediate hosts, and adaptive specificity of S. solidus to intermediate host genotype.

Nutritional status and gene expression along the somatotropic axis in roach (Rutilus rutilus) infected with the tapeworm Ligula intestinalis.

The tapeworm Ligula intestinalis inhibits gametogenesis of its fish host, the roach (Rutilus rutilus). We investigated whether L. intestinalis infection makes significant demands on nutritional resources and consequently manipulates the endocrine somatotropic axis of roach. Two groups of naturally infected and uninfected roach were studied: a field group (natural feeding) and a laboratory group (ad libitum food supply). In females, no significant impact of parasitization on storage substrates (glycogen, lipids, and protein) was detected, whereas in males, either lipid content of the liver (field group) or lipid of the muscle and glycogen of the liver (laboratory group) were slightly decreased. Except for the females of the field group, higher mRNA expression of growth hormone (gh) in the pituitary of infected fish was observed. Furthermore, the expression of hypophyseal somatolactin α and ß (slα, slß) was up-regulated in infected females of the field and laboratory group, respectively. In liver and muscle, mRNA expression of insulin-like growth factors (igf1, igf2) and igf receptor (igfr) remained either unchanged or were up-regulated with infection. Parasitization showed inconsistent effects on gh receptor 1 (ghr1) expression in liver and muscle, whereas ghr2 mRNA was mostly not influenced by infection. In general, the expression profile of genes involved in the somatotropic axis as well as the content of storage substances in infected roach did not resemble that of food-deprived fish either under natural or ad libitum feeding. In conclusion, the present study does not indicate starvation of L. intestinalis infected roach, and it is suggested that the inhibition of reproduction attenuated the nutritional demand of parasitization.

Parasite mediated homogenizing selection at the MHC in guppies.

Understanding the selective forces influencing genetic diversity is a fundamental goal of evolutionary ecology. The genes of the major histocompatibility complex (MHC) play a key role in the adaptive immune response of vertebrates and thus provide an excellent opportunity to examine the agents of selection on a functionally important gene. Here we examine the genetic architecture of the MHC class IIB genes in 10 wild populations of guppies (Poecilia reticulata) in Northern Trinidad. We have previously shown that these populations are significantly less diverged at the class IIB locus than expected based on neutral (microsatellite) loci. We now survey infection by Gyrodactylus turnbulli and G. bullatarudis, common parasitic worms that infect guppies, as a potential agent of homogenizing selection. We used a genetic algorithm to partition both additive and non-additive genetic effects of the five most common MHC allele types as well as a rare allele category. Although we found no evidence for non-additive effects, across the populations we found that one allele type (the a-type) had a significant negative additive effect on parasite load. Thus, individuals who had more copies of the a-type allele were infected with fewer gyrodactylus than individuals with fewer copies of the allele. These results not only link parasite infection with MHC genotype, they provide a mechanism of homogenizing selection across these otherwise disparate populations.

Dynamics of hepatic stellate cells, collagen types I and III synthesis and gene expression of selected cytokines during hepatic fibrogenesis following Mesocestoides vogae (Cestoda) infection in mice.

In the present study, the relationship between progression of Mesocestoides vogae infection in the liver of mice, the accumulation rate of collagen types I and III, gene expression of fibrogenic factors and cytokines was examined within 6weeks p.i. Due to asexual multiplication, the total number of larvae in the liver increased considerably and 63.4% were found in collagen capsules on day 42 p.i. Intense staining for both collagens was recorded in the activated hepatic stellate cells (HSCs) throughout the period of this study in the inflammatory lesions. With progressing infection, cellular expression of both collagens was confined to the flat cells, myofibroblasts, which were scattered among collagen fibres in parenchymal lesions and capsules. Collagen-positive areas mirrored immunostaining of alpha-smooth muscle actin (alpha-SMA) in HSCs and myofibroblasts. Gene expression of both collagens increased rapidly within 14days p.i. and their expression pattern resembled that for pro-fibrotic cytokine transforming growth factor (TGF)-beta1 and alpha-SMA protein. IL-10 cytokine expression was up-regulated following day 14 p.i. and that of IL-13 was up-regulated early p.i., then transcription elevated gradually mirroring the activity of other pro-fibrotic markers. In contrast, transcription activity of TNF-alpha and IFN-gamma was elevated shortly after infection, followed by the partial down-regulation of gene expression, indicating the lack of larval killing, enhanced granulomatous inflammation and the perpetuation of hepatic fibrosis. Histomorphometric analysis of the parenchymal fibrous lesions, surface areas of larvae surrounded with the inflammatory infiltrates and surface areas of developing or mature larva-containing granulomas, correlated with the proportion of free and encapsulated larvae, immunostaining and gene expression patterns of collagens and pro-fibrotic markers. At a later stage of infection (day 28 p.i. onwards) collagen I-positive areas occupied a greater surface area and formed mature larval capsules and scars in the liver. In contrast, collagen III was less abundant and was localised mainly in the fibrous lesions in damaged parenchyma, suggesting their specific up-regulation as the part of host-protecting and tissue-healing responses.

Geography and host specificity: two forces behind the genetic structure of the freshwater fish parasite Ligula intestinalis (Cestoda: Diphyllobothriidae).

Parasite species with global distributions and complex life cycles offer a rare opportunity to study alternative mechanisms of speciation and evolution in a single model. Here, genealogy and genetic structure, with respect to geography and fish host preference, have been analyzed for Ligula intestinalis, a tapeworm affecting freshwater fish. The data analyzed consisted of 109 tapeworms sampled from 13 fish host species in 18 different localities on a macrogeographic scale. Two mitochondrial genes, cytochrome oxidase subunit I and cytochrome B, and the nuclear sequence of intergenic transcribed spacer 2 (ITS2) were used for the genetic reconstruction. Different evolutionary patterns were found at the local and at the global geographic scales. On a local scale, the flat genetic structure was mainly attributed to contiguous range expansion. Migrating birds are the most likely cause of the homogenisation of the whole population, preventing the creation of significant genetic barriers. By contrast, on a global scale, genetically distant and well-separated clusters are present in different geographic areas. Reproductive isolation was found even between clades living in sympatry and infecting the same definitive host, suggesting the existence of efficient biologically determined genetic barriers, and thus possibly separate species. Although the ITS2 sequences were found to display considerable intragenomic variability, their relationships were generally in good agreement with the topology derived from mitochondrial genes.

Identification of an expressed gene in Dipylidium caninum.

Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.

Genetic influences upon eosinophilia and resistance in mice infected with Mesocestoides corti.

The genetic influences upon host variation in eosinophilia and resistance to helminth infection, and the relationship between these parameters, was investigated in 9 inbred and 3 hybrid strains of mice infected with Mesocestoides corti. Blood, bone marrow, spleen and peritoneal fluid eosinophilia were far higher in SJL mice than in any other inbred strain. SWR, NIH, C3H and BALB/c mice were high responders to M. corti whereas CBA and 3 congenic strains sharing the B10 background (C57BL/10, B10.S, B10.G) were low responders. Some of the genes for high eosinophil responsiveness appeared to be dominant, as F1 hybrids from high and low response parental strains were intermediate to high in response to infection. SJL and NIH strains were highly susceptible to infection with M. corti, larval burdens at 21 days after infection with 100 tetrathyridia being considerably higher (greater than 1000) than all other strains. BALB/c (congruent to 700 larvae) were designated susceptible, SWR (greater than 400 larvae) were resistant and the B10 congenics (less than 400 larvae) were highly resistant. Genes influencing resistance also appeared to be dominant, as F1 hybrids between resistant and susceptible parental strains were intermediate to resistant on infection. The overall response patterns indicate a direct correlation between susceptibility to infection and high eosinophil responsiveness, but this relationship is not consistent in all strains.

[The population and genetic relationships in the bream-Digramma interrupta system (pl.) (Cestoda: Ligulidae)]. / Izuchenie populiatsionno-geneticheskikh vzaimootnoshenii v sisteme leshch--Digramma interrupta (pl.) (Cestoda: Ligulidae).

It was established that host individuals, characterized by a high stability of development, play the main part in the functioning of bream-D. interrupta system. Heterozygous breams turned out to be more stable to the effect of pleurocercoids than homozygous ones. Genetically determined characters of bream infection with pleurocercoids of D. interrupta have been revealed. Mechanism of population genetic relationships in this parasitic system is discussed.