Project description:Marker-assisted selective breeding of fish with higher levels of resistance towards specific pathogens has shown successful, but. However, the impact of host genotype on multiple pathogen infections are is still poorly investigated. This study examined the resistance in rainbow trout (Oncorhynchus mykis) towards infection with the eye fluke Diplostomum pseudospathaceum. We used genetically selected rainbow trout, carrying SNPs associated with resistance towards the parasitic ciliate Ichthyophthirius multifiliis, and exposed the fish to eye fluke cercariae. We showed that fish partly resistant to I. multifiliis were more susceptible to eye fluke invasion. Expression The expression of immune relevant genes (encoding innate and adaptive factors) was also affected as these genotypes responded less strongly to a secondary fluke infection. The complexity of genome architecture in disease resistance towards multiple pathogens is discussed. A total of 200 rainbow trout (body weight 14.3-17.7 g, body length 10.2-11.5 cm) were used for the study. Two groups of rainbow trout with high (QTL fish) and low (non-QTL fish) frequency of SNPs associated with I. multifiliis resistance, were hatched from eyed eggs at the disease free recirculated Bornholm Salmon Hatchery, Nexø, Denmark and subsequently reared to the fingerling stage. For this purpose, the first group (QTL-fish) was produced by using sperm from three male genotyped parents carrying SNPs AX-89947214 (Omy17) and AX-89960822 (Omy16), and the other group (non-QTL fish) was produced by using sperm from three other male parents negative for these SNPs. In both cases, sperm was used to fertilize a common pool of eggs stripped from a total of 30 outbred females. Processes of hatching and subsequent rearing of fry to the fingerling stage did not differ between groups of QTL fish and non-QTL fish. From each group (QTL and non-QTL fish) we randomly gathered 100 rainbow trout and transported them (3 h duration) from the hatchery to the infection facility at the University of Copenhagen. Fish were then accommodated and acclimatized 14 d in identical aerated glass tanks with internal biofilters (25 fish per 60 L water, total tank volume 80 L), which were placed in a temperature temperature-controlled room (water temperature constant at 12°C, pH 7.6). We used 30% water change per day to maintain ammonia levels below 0.25. Fish were fed by pelleted feed (1% of fish biomass per day). All fish were genotyped with respect to the two relevant QTLs, one on chomosome 16 and one on chromosome 17. Fish being double heterozugous were excluded from qPCR analysis.

Project description:Parasite-mediated selection is considered one of the potential mechanisms contributing to the coexistence of asexual-sexual complexes. Gibel carp (Carassius gibelio), an invasive fish species in Europe, often forms populations composed of gynogenetic and sexual specimens. In the present study, experimental infection was induced in gynogenetic and sexual gibel carp using eye-fluke Diplostomum pseudospathaceum (Trematoda), and the transcriptome profile of the spleen as a major immune organ in fish was analyzed to reveal the differentially expressed immunity-associated genes related to D. pseudospathaceum infection differing between gynogenetic and sexual gibel carp. High parasite infection was found in gynogenetic fish when compared to genetically diverse sexuals. Although metacercariae of D. pseudospathaceum are situated in an immune-privileged organ, our results show that eye trematodes may induce a host immune response. We found differential gene expression induced by eye-fluke infection, with various impacts on gynogenetic and sexual hosts, documenting for the majority of DEGs upregulation in sexuals, and downregulation in asexuals. Differences in gene regulation between gynogenetic and sexual gibel carp were evidenced in many immunity-associated genes. GO analyses revealed the importance of genes assigned to the GO terms: immune function, the Notch signaling pathway, MAP kinase tyrosine/threonine/phosphatase activity, and chemokine receptor activity. KEGG analyses revealed the importance of the genes involved in 12 immunity-associated pathways – specifically, FoxO signaling, adipocytokine signaling, TGF-beta signaling, apoptosis, Notch signaling, C-type lectin receptor signaling, efferocytosis, intestinal immune network for IgA production, insulin signaling, virion - human immunodeficiency virus, Toll-like receptor signaling, and phosphatidylinositol signaling system. Our study indicates the limited potential of asexual fish to cope with higher parasite infection (likely a loss of capacity to induce an effective immune response) and highlights the important role of molecular mechanisms associated with immunity for the coexistence of gynogenetic and sexual gibel carp, potentially contributing to its invasiveness.

Project description:Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data.Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Štúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene.Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene.This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.

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Diplostomum spathaceum

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