Single-cell transcriptional analysis reveals ILC-like cells in zebrafish.

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates are poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and after immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient zebrafish that lack T and B cells, and discovered populations of ILC-like cells. We uncovered a rorc-positive subset of ILCs that could express cytokines associated with type 1, 2, and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed il22 and tnfa after exposure to inactivated bacteria or il13 after exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.The study was supported by Cancer Research UK grant number C45041/A14953 (to A.C. and E.I.A.), European Research Council project 677501 – ZF_Blood (to A.C. and P.M.S.), EMBO Long-Term Fellowship ALTF-807-2015 (to P.P.H), ANR grant 17-CE15-0017-01 – ZF-ILC (to P.P.H) and ANR-16-CE20-0002-03 (to J.-P.L), H2020-MSCA-IF-2015 grant 708128 – ZF-ILC (to P.P.H), ANR-10-LABX-73 (‘revive’ to P. Herbomel) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Resistance to a Rhabdovirus (VHSV) in Rainbow Trout: Identification of a Major QTL Related to Innate Mechanisms

Chantier qualité GAHealth control is a major issue in animal breeding and a better knowledge of the genetic bases of resistance to diseases is needed in farm animals including fish. The detection of quantitative trait loci (QTL) will help uncovering the genetic architecture of important traits and understanding the mechanisms involved in resistance to pathogens. We report here the detection of QTL for resistance to Viral Haemorrhagic Septicaemia Virus (VHSV), a major threat for European aquaculture industry. Two induced mitogynogenetic doubled haploid F2 rainbow trout (Oncorhynchus mykiss) families were used. These families combined the genome of susceptible and resistant F0 breeders and contained only fully homozygous individuals. For phenotyping, fish survival after an immersion challenge with the virus was recorded, as well as in vitro virus replication on fin explants. A bidirectional selective genotyping strategy identified seven QTL associated to survival. One of those QTL was significant at the genome-wide level and largely explained both survival and viral replication in fin explants in the different families of the design (up to 65% and 49% of phenotypic variance explained respectively). These results evidence the key role of innate defence in resistance to the virus and pave the way for the identification of the gene(s) responsible for resistance. The identification of a major QTL also opens appealing perspectives for selective breeding of fish with improved resistance

Advancing fish breeding in aquaculture through genome functional annotation

Genomics is increasingly applied in breeding programmes for farmed fish and shellfish species around the world. However, current applications do not include information on genome functional activity, which can enhance opportunities to predict relationships between genotypes and phenotypes and hence increase the accuracy of selection. Here, we review prospects for improving aquaculture breeding practises through the uptake of functional genomics data in light of the EU Horizon 2020 project AQUA-FAANG: ‘Advancing European Aquaculture by Genome Functional Annotation’. This consortium targeted the six major farmed fish species in European aquaculture, producing thousands of functional genomic datasets from samples representing embryos to mature adults of both sexes, and following immunological stimulation. This data was used to catalogue functional activity across the genome of each species, revealing transcribed regions, distinct chromatin states and regulatory elements impacting gene expression. These functional annotations were shared as open data through the Ensembl genome browser using the latest reference genomes for each species. AQUA-FAANG data offers novel opportunities to identify and prioritize causative genetic variants responsible for diverse traits including disease resistance, which can be exploited to enhance selective breeding. Such knowledge and associated resources have the potential to improve sustainability and boost production in aquaculture by accelerating genetic gain for health and robustness to infection, whilst reducing the requirement for animal testing. We further outline directions to advance and leverage genome functional annotation beyond the AQUA-FAANG project. Given the diversity of aquaculture sectors and businesses, the incorporation of functional genomic information into breeding decisions will depend on technological readiness level and scale of operation, with cost-benefit analysis necessary to determine the most profitable approach for each species and production system

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses

Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM) within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system

Transcriptional Responses of Resistant and Susceptible Fish Clones to the Bacterial Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a bacterial species that represents one of the most important pathogens for aquaculture worldwide, especially for salmonids. To gain insights into the genetic basis of the natural resistance to F. psychrophilum, we selected homozygous clones of rainbow trout with contrasted susceptibility to the infection. We compared the transcriptional response to the bacteria in the pronephros of a susceptible and a resistant line by micro-array analysis five days after infection. While the basal transcriptome of healthy fish was significantly different in the resistant and susceptible lines, the transcriptome modifications induced by the bacteria involved essentially the same genes and pathways. The response to F. psychrophilum involved antimicrobial peptides, complement, and a number of enzymes and chemokines. The matrix metalloproteases mmp9 and mmp13 were among the most highly induced genes in both genetic backgrounds. Key genes of both pro- and anti-inflammatory response such as IL1 and IL10, were up-regulated with a greater magnitude in susceptible animals where the bacterial load was also much higher. While higher resistance to F. psychrophilum does not seem to be based on extensive differences in the orientation of the immune response, several genes including complement C3 showed stronger induction in the resistant fish. They may be important for the variation of susceptibility to the infection

CXCL8 Chemokines in Teleost Fish: Two Lineages with Distinct Expression Profiles during Early Phases of Inflammation

Contains fulltext : 126584.pdf (publisher's version ) (Open Access

Metatranscriptomics and Pyrosequencing Facilitate Discovery of Potential Viral Natural Enemies of the Invasive Caribbean Crazy Ant, Nylanderia pubens

BACKGROUND: Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens. METHODOLOGY AND PRINCIPAL FINDINGS: Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens. CONCLUSIONS: Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest

Expression of the immunoglobulin lambda locus in the mouse

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Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout

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