Genome-wide alternative splicing profile in the posterior kidney of brown trout (Salmo trutta) during proliferative kidney disease.

BACKGROUND: The cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD. RESULTS: RNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value < 0.05) were identified from 19,722 alternatively spliced events. Among the DEAS genes, the least and most abundant alternative splicing types were alternative 5' splice site (5.23%) and exon skipping (70.59%), respectively. The DEAS genes were significantly enriched for sodium-potassium transporter activity and ion homeostasis (ahcyl1, atp1a3a, atp1a1a.1, and atp1a1a.5). The protein-protein interaction network analysis enriched two local network clusters namely cation transporting ATPase C-terminus and Sodium/potassium ATPase beta chain cluster, and mixed inclusion of Ion homeostasis and EF-hand domain cluster. Furthermore, the human disease-related salmonella infection pathway was significantly enriched in the protein-protein interaction network. CONCLUSION: This study provides the first baseline information about alternative splicing in brown trout during PKD. The generated data lay a foundation for further functional molecular studies in PKD - brown trout infection model. The information generated from the present study can help to develop therapeutic strategies for PKD in the future.

Genome-Wide Association Study Using Whole-Genome Sequence Data for Fertility, Health Indicator, and Endoparasite Infection Traits in German Black Pied Cattle.

This genome-wide association study (GWAS) aimed to identify sequence variants (SVs) and candidate genes associated with fertility and health in endangered German Black Pied cattle (DSN) based on whole-genome sequence (WGS) data. We used 304 sequenced DSN cattle for the imputation of 1797 genotyped DSN to WGS. The final dataset included 11,413,456 SVs of 1886 cows. Cow traits were calving-to-first service interval (CTFS), non-return after 56 days (NR56), somatic cell score (SCS), fat-to-protein ratio (FPR), and three pre-corrected endoparasite infection traits. We identified 40 SVs above the genome-wide significance and suggestive threshold associated with CTFS and NR56, and three important potential candidate genes (ARHGAP21, MARCH11, and ZNF462). For SCS, most associations were observed on BTA 25. The GWAS revealed 61 SVs, a cluster of 10 candidate genes on BTA 13, and 7 pathways for FPR, including key mediators involved in milk fat synthesis. The strongest associations for gastrointestinal nematode and Dictyocaulus viviparus infections were detected on BTA 8 and 24, respectively. For Fasciola hepatica infections, the strongest associated SVs were located on BTA 4 and 7. We detected 200 genes for endoparasite infection traits, related to 16 pathways involved in host immune response during infection.

Depletion of MHC supertype during domestication can compromise immunocompetence.

The major histocompatibility complex (MHC) plays an important role in infectious disease resistance. The presence of certain MHC alleles and functionally similar groups of MHC alleles (i.e., supertypes) has been associated with resistance to particular parasite species. Farmed and domesticated fish stocks are often depleted in their MHC alleles and supertype diversity, possibly as a consequence of artificial selection for desirable traits, inbreeding (loss of heterozygosity), genetic drift (loss of allelic diversity) and/or reduced parasite biodiversity. Here we quantify the effects of depletion of MHC class II genotype and supertype variation on resistance to the parasite Gyrodactylus turnbulli in guppies (Poecilia reticulata). Compared to the descendants of wild-caught guppies, ornamental fish had a significantly reduced MHC variation (i.e., the numbers of MHC alleles and supertypes per individual, and per population). In addition, ornamental fish were significantly more susceptible to G. turnbulli infections, accumulating peak intensity 10 times higher than that of their wildtype counterparts. Four out of 13 supertypes were associated with a significantly reduced parasite load, and the presence of some supertypes had a dramatic effect on the intensity of infection. Remarkably, the ornamental and wildtype fish differed in the supertypes that were associated with parasite resistance. Analysis with a genetic algorithm showed that resistance-conferring supertypes of the wildtype and ornamental fish shared two unique amino acids in the peptide-binding region of the MHC that were not found in any other alleles. These data show that the supertype demarcation captures some, but not all, of the variation in the immune function of the alleles. This study highlights the importance of managing functional MHC diversity in livestock, and suggests there might be some immunological redundancy among MHC supertypes.

Modulation of local and systemic immune responses in brown trout (Salmo trutta) following exposure to Myxobolus cerebralis.

Myxobolus cerebralis, the etiological agent of Whirling Disease (WD), is a freshwater myxozoan parasite with considerable economic and ecological relevance for salmonids. There are differences in disease susceptibility between species and strains of salmonids. Recently, we have reported that the suppressor of cytokine signaling SOCS1 and SOCS3 are key in modulating rainbow trout (Oncorhynchus mykiss) immune responses and that resistant fish apparently exhibit effective Th17 cell response after exposure to M. cerebralis. It is unclear whether such molecules and pathways are also involved in the immune response of M. cerebralis infected brown trout (Salmo trutta). Hence, this study aimed to explore their role during immune modulation in infected brown trout, which is considered resistant to this parasite. Fish were exposed to the triactinomyxon (TAM) stages of M. cerebralis and quantitative real-time PCR (RT-qPCR) was carried out to examine local (caudal fin) and systemic (head kidney, spleen) immune transcriptional changes associated with WD over time in infected and control fish. All of the immune genes in the three tissues studied were differentially expressed in infected fish at multiple time points. Brown trout reduced the parasite load and demonstrated effective immune responses, likely by keeping pro-inflammatory and anti-inflammatory cytokines in balance whilst stimulating efficient Th17-mediated immunity. This study increases knowledge on the brown trout immune response to M. cerebralis and helps us to understand the underlying mechanisms of WD resistance.

Identification of variants associated with hard clam, Mercenaria mercenaria, resistance to Quahog Parasite Unknown disease.

Severe losses in aquacultured and wild hard clam (Mercenaria mercenaria) stocks have been previously reported in the northeastern United States due to a protistan parasite called QPX (Quahog Parasite Unknown). Previous work demonstrated that clam resistance to QPX is under genetic control. This study identifies single nucleotide polymorphism (SNP) associated with clam survivorship from two geographically segregated populations, both deployed in an enzootic site. The analysis contrasted samples collected before and after undergoing QPX-related mortalities and relied on a robust draft clam genome assembly. ~200 genes displayed significant variant enrichment at each sampling point in both populations, including 18 genes shared between both populations. Markers from both populations were identified in genes related to apoptosis pathways, protein-protein interaction, receptors, and signaling. This research begins to identify genetic markers associated with clam resistance to QPX disease, leading the way for the development of resistant clam stocks through marker-assisted selection.

Identification and Expression Profiling of Toll-Like Receptors of Brown Trout (Salmo trutta) during Proliferative Kidney Disease.

Proliferative kidney disease is an emerging disease among salmonids in Europe and North America caused by the myxozoan parasite Tetracapsuloides bryosalmonae. The decline of endemic brown trout (Salmo trutta) in the Alpine streams of Europe is fostered by T. bryosalmonae infection. Toll-like receptors (TLRs) are a family of pattern recognition receptors that acts as sentinels of the immune system against the invading pathogens. However, little is known about the TLRs' response in salmonids against the myxozoan infection. In the present study, we identified and evaluated TLR1, TLR19, and TLR13-like genes of brown trout using data-mining and phylogenetic analysis. The expression pattern of TLRs was examined in the posterior kidney of brown trout infected with T. bryosalmonae at various time points. Typical Toll/interleukin-1 receptor protein domain was found in all tested TLRs. However, TLR13-like chr2 had a short amino acid sequence with no LRR domain. Phylogenetic analysis illustrated that TLR orthologs are conserved across vertebrates. Similarly, a conserved synteny gene block arrangement was observed in the case of TLR1 and TLR19 across fish species. Interestingly, all tested TLRs showed their maximal relative expression from 6 to 10 weeks post-exposure to the parasite. Our results suggest that these TLRs may play an important role in the innate defense mechanism of brown trout against the invading T. bryosalmonae.

Effects of Enteromyxum spp. (Myxozoa) infection in the regulation of intestinal E-cadherin: Turbot against gilthead sea bream.

Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host-parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host-parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture.

Cumulative effects of cadmium and natural stressors (temperature and parasite infection) on molecular and biochemical responses of juvenile rainbow trout.

The simultaneous presence of natural and anthropogenic stressors in aquatic ecosystems can challenge the identification of factors causing decline in fish populations. These stressors include chemical mixtures and natural abiotic and biotic factors such as water temperature and parasitism. Effects of cumulative stressors may vary from antagonism to synergism at the organismal or population levels and may not be predicted from exposure to individual stressors. This study aimed to evaluate the combined effects of chronic exposure to cadmium (Cd) and elevated water temperature (23 °C) or parasite infection in juvenile rainbow trout (Oncorhynchus mykiss) using a multi-level biological approach, including RNA-sequencing. Fish were exposed to diet-borne Cd (6 µg Cd/g wet feed), individually and in combination with thermal (23 °C) or parasitic stressors, for 28 days. The parasite challenge consisted of a single exposure to glochidia (larvae) of the freshwater mussel (Strophitus undulatus), which encysts in fish gills, fins and skin. Results indicated lower fish length, weight, and relative growth rate in fish exposed to a higher water temperature (23 °C). Body condition and hepatosomatic index of trout were, however, higher in the 23 °C temperature treatment compared to the control fish kept at 15 °C. Exposure to thermal stress or parasitism did not influence tissue Cd bioaccumulation. More than 700 genes were differentially transcribed in fish exposed to the individual thermal stress treatment. However, neither Cd exposure nor parasite infection affected the number of differentially transcribed genes, compared to controls. The highest number of differentially transcribed genes (969 genes) was observed in trout exposed to combined Cd and high temperature stressors; these genes were mainly related to stress response, protein folding, calcium metabolism, bone growth, energy metabolism, and immune system; functions overlapped with responses found in fish solely exposed to higher water temperature. Only 40 genes were differentially transcribed when fish were exposed to Cd and glochidia and were related to the immune system, apoptosis process, energy metabolism and malignant tumor. These results suggest that dietary Cd may exacerbate the temperature stress and, to a lesser extent, parasitic infection stress on trout transcriptomic responses. Changes in the concentrations of liver ethoxyresorufin-o-deethylase, heat shock protein 70 and thiobarbituric acid reactive substances coupled to changes in the activities of cellular glutathione S-transferase and glucose-6-phosphate dehydrogenase were also observed at the cellular level. This study may help understand effects of freshwater fish exposure to cumulative stressors in a changing environment.

A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a 'One Health' context.

Studying the epidemiology of schistosomiasis-the most prevalent gastropod-borne human disease and an economic burden for the livestock industry-relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.

Disease mortality in domesticated animals is predicted by host evolutionary relationships.

Infectious diseases of domesticated animals impact human well-being via food insecurity, loss of livelihoods, and human infections. While much research has focused on parasites that infect single host species, most parasites of domesticated mammals infect multiple species. The impact of multihost parasites varies across hosts; some rarely result in death, whereas others are nearly always fatal. Despite their high ecological and societal costs, we currently lack theory for predicting the lethality of multihost parasites. Here, using a global dataset of >4,000 case-fatality rates for 65 infectious diseases (caused by microparasites and macroparasites) and 12 domesticated host species, we show that the average evolutionary distance from an infected host to other mammal host species is a strong predictor of disease-induced mortality. We find that as parasites infect species outside of their documented phylogenetic host range, they are more likely to result in lethal infections, with the odds of death doubling for each additional 10 million years of evolutionary distance. Our results for domesticated animal diseases reveal patterns in the evolution of highly lethal parasites that are difficult to observe in the wild and further suggest that the severity of infectious diseases may be predicted from evolutionary relationships among hosts.

Survival of Whirling-Disease-Resistant Rainbow Trout Fry in the Wild: A Comparison of Two Strains.

Introduced pathogens can affect fish populations, and three main factors affect disease occurrence: the environment, host, and pathogen. Manipulating at least one of these factors is necessary for controlling disease. Myxobolus cerebralis, the parasite responsible for salmonid whirling disease, became established in Colorado during the 1990s and caused significant declines in wild Rainbow Trout Oncorhynchus mykiss populations. Attempts to re-establish Rainbow Trout have focused on manipulating salmonid host resistance. A Rainbow Trout strain known as GR × CRR was developed for stocking in Colorado by crossing a whirling-disease-resistant strain known as the German Rainbow Trout (GR) with the Colorado River Rainbow Trout (CRR). The GR × CRR fish exhibit resistance similar to that shown by GR, and survival and reproduction were expected to be similar to those of CRR. One disadvantage of stocking GR × CRR is that outcrossing and backcrossing could decrease resistance, and laboratory studies have indicated that this can occur. A potential disadvantage of stocking pure GR is lower survival due to domestication. To compare fry survival between the strains, a field experiment was conducted in 1.6-km reaches of nine Colorado streams. Each stream was stocked in August 2014 with 5,000 GR × CRR and 5,000 GR individuals. In October 2014, April 2015, and August 2015, apparent survival was assessed. Two laboratory predation experiments were also conducted. The field experiment revealed that short-term apparent survival was influenced by stream, and growth rate was influenced by strain and stream. However, after 12 months, there was no difference in apparent survival or growth rate between the GR and GR × CRR strains. Laboratory experiments showed that survival did not differ between the strains when confronted with Brown Trout Salmo trutta predation. Our results indicate that the GR strain is a viable option for stocking in streams where M. cerebralis is enzootic. Further evaluation is needed to determine whether GR fish will survive to maturity and reproduce.

Biodiversity and host-parasite cophylogeny of Sphaerospora (sensu stricto) (Cnidaria: Myxozoa).

BACKGROUND: Myxozoa are extremely diverse microscopic parasites belonging to the Cnidaria. Their life-cycles alternate between vertebrate and invertebrate hosts, predominantly in aquatic habitats. Members of the phylogenetically well-defined Sphaerospora (sensu stricto) clade predominantly infect the urinary system of marine and freshwater fishes and amphibians. Sphaerosporids are extraordinary due to their extremely long and unique insertions in the variable regions of their 18S and 28S rDNA genes and due to the formation of motile proliferative stages in the hosts' blood. To date, DNA sequences of only 19 species have been obtained and information on the patterns responsible for their phylogenetic clustering is limited. METHODS: We screened 549 fish kidney samples from fish of various geographical locations, mainly in central Europe, to investigate sphaerosporid biodiversity microscopically and by 18S rDNA sequences. We performed multiple phylogenetic analyses to explore phylogenetic relationships and evolutionary trends within the Sphaerospora (s.s.) clade, by matching host and habitat features to the resultant 18S rDNA trees. The apparent co-clustering of species from related fish hosts inspired us to further investigate host-parasite co-diversification, using tree-based (CoRE-PA) and distance-based (ParaFit) methods. RESULTS: Our study considerably increased the number of 18S rDNA sequence data for Sphaerospora (s.s.) by sequencing 17 new taxa. Eight new species are described and one species (Sphaerospora diminuta Li & Desser, 1985) is redescribed, accompanied by sufficient morphological data. Phylogenetic analyses showed that sphaerosporids cluster according to their vertebrate host order and habitat, but not according to geography. Cophylogenetic analyses revealed a significant congruence between the phylogenetic trees of sphaerosporids and of their vertebrate hosts and identified Cypriniformes as a host group of multiple parasite lineages and with high parasite diversity. CONCLUSIONS: This study significantly contributed to our knowledge of the biodiversity and evolutionary history of the members of the Sphaerospora (s.s.) clade. The presence of two separate phylogenetic lineages likely indicates independent historical host entries, and the remarkable overlap of the larger clade with vertebrate phylogeny suggests important coevolutionary adaptations. Hyperdiversification of sphaerosporids in cypriniform hosts, which have undergone considerable radiations themselves, points to host-driven diversification.

Comparison of Sampling and Detection Methods for Chinook Salmon and Steelhead Naturally Infected with Myxobolus cerebralis.

Myxobolus cerebralis (Mc) is a myxozoan parasite causing whirling disease in hatchery- and natural-origin salmonids. To minimize spread of this parasite and the incidence of its associated disease, fish health professionals routinely screen fish for Mc before stocking or moving the fish to Mc-free waters. Sample collection for Mc traditionally entails lethal sampling of cranial tissue followed by pepsin-trypsin digest (PTD) and screening of the sample for mature myxobolid myxospores (PTD method); however, nonlethal sampling methods would be advantageous in some circumstances, such as when dealing with rare or otherwise valuable fish. Accordingly, we compared Mc detections in cranial cartilage by using the PTD method with PCR assays of fin biopsies collected from juvenile Chinook Salmon Oncorhynchus tshawytscha and adult steelhead O. mykiss. Cranial samples were also analyzed using PCR methods for comparative purposes. Results indicated that Mc could be detected by PCR in fin clips, but the results generated by this approach differed significantly from those associated with PTD- and/or PCR-based analysis of cranial cartilage samples. Polymerase chain reaction-based analysis-of individual head samples and head digest pools in both species as well as fins in steelhead-yielded more positive detections than PTD analysis alone. The PCR-based analysis of head and fin tissues yielded different Mc detection rates in both species, but the nature of the detection disparity varied depending on the species and/or life stage of the fish. We conclude that for lethal cranial samples, neither PTD nor PCR should be used alone, but using these techniques in concert may provide the most complete and accurate estimation of Mc presence in a group of salmonids. If imperiled or highly valuable fish are in question, nonlethal fin samples may be used to generate some information regarding Mc status, with the understanding that parasite DNA detections do not necessarily signify mature infections or disease.

Genetic correlations between endo-parasite phenotypes and economically important traits in dairy and beef cattle.

Parasitic diseases have economic consequences in cattle production systems. Although breeding for parasite resistance can complement current control practices to reduce the prevalence globally, there is little knowledge of the implications of such a strategy on other performance traits. Records on individual animal antibody responses to Fasciola hepatica, Ostertagia ostertagi, and Neospora caninum were available from cows in 68 dairy herds (study herds); national abattoir data on F. hepatica-damaged livers were also available from dairy and beef cattle. After data edits, 9,271 dairy cows remained in the study herd dataset, whereas 19,542 dairy cows and 68,048 young dairy and beef animals had a record for the presence or absence of F. hepatica-damaged liver in the national dataset. Milk, reproductive, and carcass phenotypes were also available for a proportion of these animals as well as their contemporaries. Linear mixed models were used to estimate variance components of antibody responses to the three parasites; covariance components were estimated between the parasite phenotypes and economically important traits. Heritability of antibody responses to the different parasites, when treated as a continuous trait, ranged from 0.07 (O. ostertagi) to 0.13 (F. hepatica), whereas the coefficient of genetic variation ranged from 4% (O. ostertagi) to 20% (F. hepatica). The antibody response to N. caninum was genetically correlated with the antibody response to both F. hepatica (-0.29) and O. ostertagi (-0.67); a moderately positive genetic correlation existed between the antibody response to F. hepatica and O. ostertagi (0.66). Genetic correlations between the parasite phenotypes and the milk production traits were all close to zero (-0.14 to 0.10), as were the genetic correlations between F. hepatica-damaged livers and the carcass traits of carcass weight, conformation, and fat score evaluated in cows and young animals (0.00 to 0.16). The genetic correlation between F. hepatica-damaged livers in cows and milk somatic cell score was 0.32 (SE = 0.20). Antibody responses to F. hepatica and O. ostertagi had favorable genetic correlations with fertility traits, but conversely, antibody response to N. caninum and F. hepatica-damaged livers were unfavorably genetically correlated with fertility. This study provides the necessary information to undertake national multitrait genetic evaluations for parasite phenotypes.

Telomere length and antioxidant defense associate with parasite-induced retarded growth in wild brown trout.

Early growth conditions can have profound impacts on individuals' development, growth and physiology, with subsequent long-term consequences for individuals' fitness and life expectancy. Telomere length (TL) has been suggested to indicate both individual fitness and life expectancy in wide range of species, as the telomere attrition rate at early age can be accelerated due to exposure to various stressors, including parasites and inflammatory diseases, which increase production of reactive oxygen species (ROS) and influence antioxidant (AO) levels. We investigated impacts of Tetracapsuloides bryosalmonae infection, a causative agent of proliferative kidney disease (PKD), on AO status and TL in a natural population of juvenile brown trout (Salmo trutta). The fish with higher parasite load showed more severe kidney hyperplasia, anemia and smaller body size compared to less parasitized fish. Furthermore, fish with severe PKD symptoms had lower SOD-, CAT- and GST activity than fish with milder kidney hyperplasia. However, parasite load was not directly correlated either with AOs or with TL. Smaller fish showed shorter TLs, potentially reflecting lower individual quality. The fish, which were less sensitive to parasite-induced impaired growth, quantified as parasite load-adjusted fork length, showed also longer TLs, lower GR- and GST activity and less GSHtot compared to more sensitive fish. These results provide novel knowledge about the impacts of the PKD in brown trout at the molecular level and support the idea that TL may reflect individual quality and ability to cope with parasitic infections.

A RNAi-based therapeutic proof of concept targets salmonid whirling disease in vivo.

Myxobolus cerebralis is a cnidarian-myxozoan parasite that causes salmonid whirling disease. M. cerebralis alternates between two hosts: (1) a vertebrate salmonid and (2) an invertebrate oligochaete, Tubifex tubifex. There is no successful treatment for salmonid whirling disease. MyxSP-1 is a M. cerebralis serine protease implicated in whirling disease pathogenesis. We hypothesized that short-interfering RNA (siRNA)-induced RNA interference (RNAi) can silence MyxSP-1 in the invertebrate host and abrogate the M. cerebralis life cycle. This would preclude whirling disease infection in the salmonid host. To test this hypothesis, we first developed a siRNA delivery protocol in T. tubifex. Second, we determined the effective dose for siRNA treatment of M. cerebralis-infected T. tubifex. M. cerebralis-infected T. tubifex were treated with different concentrations of MyxSP-1 or negative control siRNAs (1µM, 2µM, 5µM or 7µM) at 15°C for 24h, 48h, 72h and 96h, respectively. We monitored MyxSP-1 knockdown using real-time quantitative PCR (qPCR). siRNA treatment with MyxSP-1 siRNA at 2µM concentration for 24h at 15°C showed maximum significant MyxSP-1 knockdown in T. tubifex. Third, we determined the time points in the M. cerebralis life cycle in T. tubifex at which siRNA treatment was most effective. M. cerebralis-infected T. tubifex were treated with MyxSP-1 or negative control siRNAs (2µM concentration for 24h at 15°C) at 24 hours post-infection (24hpi), 48hpi, 72hpi, 96hpi, 1 month post-infection (1mpi), 2mpi and 3mpi, respectively. We observed that siRNA treatment of T. tubifex was most effective at 1mpi, 2mpi and 3mpi. Fourth, we immersed specific-pathogen-free rainbow trout fry in water inhabited by MyxSP-1 siRNA-treated T. tubifex (at 1mpi, 2mpi and 3mpi). The salmonids did not develop whirling disease and showed significant MyxSP-1 knockdown. We also observed long-term RNAi in T. tubifex. Together these results demonstrate a novel RNAi-based therapeutic proof of concept in vivo against salmonid whirling disease.

Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation?

Hybrids and their parasite diversity represent interesting models for evolutionary ecology. The modified immune response, shifted ecology, inheritance, and maternal ancestry of hybrid host fish are supposed to affect the diversity of their parasite communities. The pattern of metazoan parasite distribution in non-congeneric cyprinids - common bream (Abramis brama) and roach (Rutilus rutilus) (species with different morphology and ecology, and harbouring different specific parasites) - and their hybrids was analysed. Four static alternative scenarios based on parasite infection levels in hybrids and parental taxa are known. The hybrid resistance scenario predicts that hybrids are more resistant than parental taxa, resulting in low parasite infection in hybrids. This scenario is principally consistent with hybrid heterosis advantage. In accordance with this prediction, metazoan parasite abundance and prevalence were higher in parental species when compared with their hybrids. Alternatively, the dynamic Red Queen scenario of infection in hybridising systems predicts parasite adaptation to common hosts. Temporal (six sampling events) and spatial (two sampling sites) aspects as possible factors influencing parasite distribution were analysed. We found no support for this hypothesis, i.e. no changes in the frequency of hybrids or their parental species and no changes in parasite infection in parental species or hybrids were found in the different time periods. The effect of maternal ancestry on infection level was evident; hybrids exhibiting common bream mtDNA were more strongly parasitized by digeneans and crustaceans than hybrids exhibiting roach mtDNA. Hybrids harboured a majority of the specific parasites of both parental species; however, the level of infection of common bream-specific parasites (especially monogeneans) in hybrids was low. Such an asymmetrical distribution of parental species-specific parasites in hybrids may suggest the limited inheritance of protective immunological mechanisms from one parental species and reveal stronger coadaptation between common bream and its specific parasites.

Maximising fitness in the face of parasites: a review of host tolerance.

Tolerance, the ability of a host to limit the negative fitness effects of a given parasite load, is now recognised as an important host defence strategy in animals. Together with resistance, the ability of a host to limit parasite load, these two host strategies represent two disparate host responses to parasites, each with different predicted evolutionary consequences: resistance is predicted to reduce parasite prevalence, whereas tolerance could be neutral towards, or increase, parasite prevalence in a population. The distinction between these two strategies might have far-reaching epidemiological consequences. Classically, a reaction norm defines host tolerance because it depicts the change in host fitness as a function of parasite load, where a shallow negative slope indicates that host fitness slowly deteriorates as parasite load increases (i.e., high tolerance). Despite the fact that tolerance was only recently acknowledged to be an important component in an animal's immune repertoire, it is frequently referenced, so our aim is to emphasise the current advances on the topic. We begin by summarising the ways in which biologists measure the two components of tolerance, parasite load and fitness, as well as the ways in which the concept has been defined (i.e., point and range tolerance). It is common to test for variation in host tolerance according to intrinsic, innate factors, where variation exists among populations, genders or genotypes. Such variation in tolerance is pervasive across animal taxa, and we briefly review some of the mechanistic bases of variation that have recently begun to be explored. Three further novel advancements in the tolerance field are the appreciation of the role of extrinsic, environmental factors on tolerance, host tolerance in multi-host-parasite systems and individual-based approaches to tolerance measures. We explore these topics using recent examples and suggest some future perspectives. It is becoming increasingly clear that an appreciation of tolerance as a defence strategy can provide significant insights into how hosts coexist with parasites.

Development of novel associations between MHC alleles and susceptibility to parasitic infections in an isolated population of an endangered mammal.

The role of pathogens in dynamics of endangered species is not fully understood, and the effect of infection often interacts with other processes affecting those species, such as fragmentation and isolation or loss of genetic variation. Small, isolated populations are prone to losing functional alleles due to demographic processes and genetic drift, which may diminish their ability to resist infection if immune genes are affected. Demographic processes may also alter the selective pressure exerted by a parasite, as they influence the rate of parasite transmission between individuals. In the present paper we studied changes in parasite infection levels and genetic variability in an isolated population of spotted suslik (Spermophillus suslicus). Over a three-year period (approx. three generations), when the population size remained relatively stable, we observed a considerable increase in parasite prevalence and infection intensity, followed by the development of novel associations between MHC DRB alleles and parasite burden. Contrary to expectations, the change in MHC allele frequency over time was not consistent with the effect of the allele - for instance, Spsu-DRB*07, associated with higher intensity of infection with a nematode Capillaria sp., increased in frequency from 11.8 to 20.2%. Yet, we found no signatures of selection in the studied loci. Our results show that an isolated, stable population may experience a sudden increase in parasitic infections, resulting in a development of novel associations between MHC alleles and parasite susceptibility/resistance, even though no signatures of selection can be found.

Can environmental change affect host/parasite-mediated speciation?

Parasitism can be a driver of species divergence and thereby significantly alter species formation processes. While we still need to better understand how parasite-mediated speciation functions, it is even less clear how this process is affected by environmental change. Both rapid and gradual changes of the environment can modify host immune responses, parasite virulence and the specificity of their interactions. They will thereby change host-parasite evolutionary trajectories and the potential for speciation in both hosts and parasites. Here, we summarise mechanisms of host-parasite interactions affecting speciation and subsequently consider their susceptibility to environmental changes. We mainly focus on the effects of temperature change and nutrient input to ecosystems as they are major environmental stressors. There is evidence for both disruptive and accelerating effects of those pressures on speciation that seem to be context-dependent. A prerequisite for parasite-driven host speciation is that parasites significantly alter the host's Darwinian fitness. This can rapidly lead to divergent selection and genetic adaptation; however, it is likely preceded by more short-term plastic and transgenerational effects. Here, we also consider how these first responses and their susceptibility to environmental changes could lead to alterations of the species formation process and may provide alternative pathways to speciation.