Interplay between a bacterial pathogen and its host in rainbow trout isogenic lines with contrasted susceptibility to cold water disease.

Infectious diseases are a major constraint on aquaculture. Genetic lines with different susceptibilities to diseases are useful models to identify resistance mechanisms to pathogens and to improve prophylaxis. Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum represents a major threat for freshwater salmonid farming worldwide. A collection of rainbow trout (Oncorhynchus mykiss) isogenic lines was previously produced from a French domestic population. Here, we compared BCWD resistance phenotypes using a subset of isogenic lines chosen for their contrasted susceptibilities to F. psychrophilum. We applied individual monitoring to document the infection process, including time-course quantification of bacteremia and innate immune response. Strikingly, BCWD resistance was correlated with a lower bacterial growth rate in blood. Several immune genes were expressed at higher levels in resistant fish regardless of infection: the Type II arginase (arg2), a marker for M2 macrophages involved in anti-inflammatory responses and tissue repair, and two Toll-like receptors (tlr2/tlr7), responsible for pathogen detection and inflammatory responses. This study highlights the importance of innate and intrinsic defense mechanisms in determining the outcome of F. psychrophilum infections, and illustrates that non-lethal time-course blood sampling for individual monitoring of bacteremia is a powerful tool to resolve within-host pathogen behavior in bacterial fish diseases.

Development of a High-Density 665 K SNP Array for Rainbow Trout Genome-Wide Genotyping.

Single nucleotide polymorphism (SNP) arrays, also named « SNP chips ¼, enable very large numbers of individuals to be genotyped at a targeted set of thousands of genome-wide identified markers. We used preexisting variant datasets from USDA, a French commercial line and 30X-coverage whole genome sequencing of INRAE isogenic lines to develop an Affymetrix 665 K SNP array (HD chip) for rainbow trout. In total, we identified 32,372,492 SNPs that were polymorphic in the USDA or INRAE databases. A subset of identified SNPs were selected for inclusion on the chip, prioritizing SNPs whose flanking sequence uniquely aligned to the Swanson reference genome, with homogenous repartition over the genome and the highest Minimum Allele Frequency in both USDA and French databases. Of the 664,531 SNPs which passed the Affymetrix quality filters and were manufactured on the HD chip, 65.3% and 60.9% passed filtering metrics and were polymorphic in two other distinct French commercial populations in which, respectively, 288 and 175 sampled fish were genotyped. Only 576,118 SNPs mapped uniquely on both Swanson and Arlee reference genomes, and 12,071 SNPs did not map at all on the Arlee reference genome. Among those 576,118 SNPs, 38,948 SNPs were kept from the commercially available medium-density 57 K SNP chip. We demonstrate the utility of the HD chip by describing the high rates of linkage disequilibrium at 2-10 kb in the rainbow trout genome in comparison to the linkage disequilibrium observed at 50-100 kb which are usual distances between markers of the medium-density chip.

Why Do Some Rainbow Trout Genotypes Grow Better With a Complete Plant-Based Diet? Transcriptomic and Physiological Analyses on Three Isogenic Lines.

Within the context of a growing aquaculture production coupled with a plateau of the production in the main components of aquafeeds (fish oil and fishmeal), recent studies have typically focused on replacing these feedstuffs with terrestrial plant ingredients for cultured carnivorous aquatic species, such as rainbow trout (Oncorhynchus mykiss). Substitution rates without adverse effects have, however, reached their limit. One potential way forward would be to take advantage of the genetic variability that exists in the salmonid population. However, to date, little is known about the underlying molecular mechanisms responsible for this genetic variability. The aim of the present research was to understand why some genotypes are better able to utilize plant-based diets devoid of marine resources. In this regard, three isogenic lines of rainbow trout (R23h, AB1h, and A22h), with similar growth when fed marine resources-based diets and which differ greatly in their responses to a plant-based diet, were fed with either a complete plant-based diet (V diet) or a marine resources-based diet (M diet) since first-feeding. Fish traits and the hepatic transcriptome of these three genotypes were compared after 5 months of feeding. First, differences in the ability to grow with the V diet observed amongst genotypes was not due to higher feed intake, but instead due to differences in feed efficiency. The comparison of transcriptome profiles revealed 575 (R23h vs. AB1h), 1,770 (R23h vs. A22h), and 2,973 (AB1h vs. A22h) probes differentially expressed amongst the three genotypes when fed the V diet. Interestingly, R23h and AB1h fish, which were the least affected by the V diet, exhibited the highest growth. These results demonstrate that these fish were able to maintain a high level of energy production and protein synthesis. Moreover, these genotypes were also able to activate pathways linked to lipid and cholesterol metabolisms, such as the biosynthesis of long-chain polyunsaturated fatty acids. Finally, as previously, immunity seems to also play an important role in the ability of fish to use the V diet, and further studies are needed to understand the mechanisms by which immunity interacts with growth.

Cutting Edge: Neutralizing Public Antibody Responses Are an Ancient Form of Defense Conserved in Fish and Mammals.

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHµ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.

Sources of variation of DNA methylation in rainbow trout: combined effects of temperature and genetic background.

Phenotypic plasticity is a key component of the ability of organisms to respond to changing environmental conditions. In this study, we aimed to study the establishment of DNA methylation marks in response to an environmental stress in rainbow trout and to assess whether these marks depend on the genetic background. The environmental stress chosen here was temperature, a known induction factor of epigenetic marks in fish. To disentangle the role of epigenetic mechanisms such as DNA methylation in generating phenotypic variations, nine rainbow trout isogenic lines with no genetic variability within a line were used. For each line, half of the eggs were incubated at standard temperature (11°C) and the other half at high temperature (16°C), from eyed-stage to hatching. In order to gain a first insight into the establishment of DNA methylation marks in response to an early temperature regime (control 11°C vs. heated 16°C), we have studied the expression of 8 dnmt3 (DNA methyltransferase) genes, potentially involved in de novo methylation, and analysed global DNA methylation in the different rainbow trout isogenic lines using LUMA (LUminometric Methylation Assay). Finally, finer investigation of genome-wide methylation patterns was performed using EpiRADseq, a reduced-representation library approach based on the ddRADseq (Double Digest Restriction Associated DNA) protocol, for six rainbow trout isogenic lines. We have demonstrated that thermal history during embryonic development alters patterns of DNA methylation, but to a greater or lesser extent depending on the genetic background.

Genetic determinism of spontaneous masculinisation in XX female rainbow trout: new insights using medium throughput genotyping and whole-genome sequencing.

Rainbow trout has a male heterogametic (XY) sex determination system controlled by a major sex-determining gene, sdY. Unexpectedly, a few phenotypically masculinised fish are regularly observed in all-female farmed trout stocks. To better understand the genetic determinism underlying spontaneous maleness in XX-rainbow trout, we recorded the phenotypic sex of 20,210 XX-rainbow trout from a French farm population at 10 and 15 months post-hatching. The overall masculinisation rate was 1.45%. We performed two genome-wide association studies (GWAS) on a subsample of 1139 individuals classified as females, intersex or males using either medium-throughput genotyping (31,811 SNPs) or whole-genome sequencing (WGS, 8.7 million SNPs). The genomic heritability of maleness ranged between 0.48 and 0.62 depending on the method and the number of SNPs used for the estimation. At the 31K SNPs level, we detected four QTL on three chromosomes (Omy1, Omy12 and Omy20). Using WGS information, we narrowed down the positions of the two QTL detected on Omy1 to 96 kb and 347 kb respectively, with the second QTL explaining up to 14% of the total genetic variance of maleness. Within this QTL, we detected three putative candidate genes, fgfa8, cyp17a1 and an uncharacterised protein (LOC110527930), which might be involved in spontaneous maleness of XX-female rainbow trout.

Combining Multiple Approaches and Models to Dissect the Genetic Architecture of Resistance to Infections in Fish.

Infectious diseases represent a major threat for the sustainable development of fish farming. Efficient vaccines are not available against all diseases, and growing antibiotics resistance limits the use of antimicrobial drugs in aquaculture. It is therefore important to understand the basis of fish natural resistance to infections to help genetic selection and to develop new approaches against infectious diseases. However, the identification of the main mechanisms determining the resistance or susceptibility of a host to a pathogenic microbe is challenging, integrating the complexity of the variation of host genetics, the variability of pathogens, and their capacity of fast evolution and adaptation. Multiple approaches have been used for this purpose: (i) genetic approaches, QTL (quantitative trait loci) mapping or GWAS (genome-wide association study) analysis, to dissect the genetic architecture of disease resistance, and (ii) transcriptomics and functional assays to link the genetic constitution of a fish to the molecular mechanisms involved in its interactions with pathogens. To date, many studies in a wide range of fish species have investigated the genetic determinism of resistance to many diseases using QTL mapping or GWAS analyses. A few of these studies pointed mainly toward adaptive mechanisms of resistance/susceptibility to infections; others pointed toward innate or intrinsic mechanisms. However, in the majority of studies, underlying mechanisms remain unknown. By comparing gene expression profiles between resistant and susceptible genetic backgrounds, transcriptomics studies have contributed to build a framework of gene pathways determining fish responsiveness to a number of pathogens. Adding functional assays to expression and genetic approaches has led to a better understanding of resistance mechanisms in some cases. The development of knock-out approaches will complement these analyses and help to validate putative candidate genes critical for resistance to infections. In this review, we highlight fish isogenic lines as a unique biological material to unravel the complexity of host response to different pathogens. In the future, combining multiple approaches will lead to a better understanding of the dynamics of interaction between the pathogen and the host immune response, and contribute to the identification of potential targets of selection for improved resistance.

Sequential Immunization With Heterologous Viruses Does Not Result in Attrition of the B Cell Memory in Rainbow Trout.

Long-term immunity is of great importance for protection against pathogens and has been extensively studied in mammals. Successive heterologous infections can affect the maintenance of immune memory, inducing attrition of T memory cells and diminishing B cell mediated protection. In fish, the basis of immune memory and the mechanisms of immunization to heterologous pathogens remain poorly understood. We sequentially immunized isogenic rainbow trout with two immunologically distinct viruses, VHSV and IPNV, either with one virus only or in combination, and analyzed the antibody responses and repertoires. Neutralizing antibodies and ELISPOT did not reveal an effect of heterologous immunization. Using a consensus read sequencing approach that incorporates unique barcodes to each cDNA molecule, we focused on the diversity expressed by selected responding VH/C combinations. We identified both public and private responses against VHSV and/or IPNV in all groups of fish. In fish immunized with two viruses, we registered no significant reduction in the persistence of the response toward the primary immunization. Similarly, the response to the second immunization was not affected by a prior vaccination to the other virus. Our data suggest that heterologous immunization does not enforce attrition of pre-existing antibody producing cells, which may impair the protection afforded by multiple successive vaccinations. These observations are potentially important to improve vaccination strategies practiced in aquaculture.

Genome-wide estimates of genetic diversity, inbreeding and effective size of experimental and commercial rainbow trout lines undergoing selective breeding.

BACKGROUND: Selective breeding is a relatively recent practice in aquaculture species compared to terrestrial livestock. Nevertheless, the genetic variability of farmed salmonid lines, which have been selected for several generations, should be assessed. Indeed, a significant decrease in genetic variability due to high selection intensity could have occurred, potentially jeopardizing the long-term genetic progress as well as the adaptive capacities of populations facing change(s) in the environment. Thus, it is important to evaluate the impact of selection practices on genetic diversity to limit future inbreeding. The current study presents an analysis of genetic diversity within and between six French rainbow trout (Oncorhynchus mykiss) experimental or commercial lines based on a medium-density single nucleotide polymorphism (SNP) chip and various molecular genetic indicators: fixation index (FST), linkage disequilibrium (LD), effective population size (Ne) and inbreeding coefficient derived from runs of homozygosity (ROH). RESULTS: Our results showed a moderate level of genetic differentiation between selected lines (FST ranging from 0.08 to 0.15). LD declined rapidly over the first 100 kb, but then remained quite high at long distances, leading to low estimates of Ne in the last generation ranging from 24 to 68 depending on the line and methodology considered. These results were consistent with inbreeding estimates that varied from 10.0% in an unselected experimental line to 19.5% in a commercial line, and which are clearly higher than corresponding estimates in ruminants or pigs. In addition, strong variations in LD and inbreeding were observed along the genome that may be due to differences in local rates of recombination or due to key genes that tended to have fixed favorable alleles for domestication or production. CONCLUSIONS: This is the first report on ROH for any aquaculture species. Inbreeding appeared to be moderate to high in the six French rainbow trout lines, due to founder effects at the start of the breeding programs, but also likely to sweepstakes reproductive success in addition to selection for the selected lines. Efficient management of inbreeding is a major goal in breeding programs to ensure that populations can adapt to future breeding objectives and SNP information can be used to manage the rate at which inbreeding builds up in the fish genome.

Correction to: Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

What could be the mechanisms of immunological memory in fish?

Vaccination is the most effective strategy to control infectious diseases in species with adaptive immunity. In human and in mouse, vaccination typically induces specific memory cells, which can mediate a fast anamnestic response upon infection by the targeted pathogen. In these species, successful vaccination induces a long-lasting protection, long after the titres of specific antibodies and the frequency of specific T cells have returned to steady state. Vaccination is also an important challenge in aquaculture, since alternative treatments are either too costly, or, in the case of antibiotics, are harmful for the environment or may result in dangerous resistances. However, the mechanisms of the long-term protection elicited by vaccines in fish remain poorly understood. Although fish possess typical B- and T-cells expressing diverse repertoires of immunoglobulins and T-cell receptors, many features of antigen specific responses are different from what is known in mouse and in human. Memory is one of the most elusive properties of fish adaptive immunity, and its basis is widely unknown. In this opinion article, we discuss the concept of immune memory in the context of the fish immunity. We illustrate the complexity of this question by discussing the results of experiments showing that protection can be passed through adoptive transfer of leukocytes from vaccinated donor fish to naive histocompatible recipients. Combined with tools developed in Targetfish and in previous projects, such as monoclonal antibodies against B- and T-cell markers, we propose that such models of protection transfer provide excellent systems to dissect the mechanisms of B- and T-cell memory in the future.

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus.

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

Regulation by Dietary Carbohydrates of Intermediary Metabolism in Liver and Muscle of Two Isogenic Lines of Rainbow Trout.

Rainbow trout (Oncorhynchus mykiss) is recognized as a typical "glucose-intolerant" fish, and the limits of dietary carbohydrate utilization have been investigated for many years. In this study, the objective was to test the molecular effects of dietary carbohydrates on intermediary metabolism in two major metabolic tissues, liver and muscle. Another objective was also to study if the response to carbohydrate intake depended on the genetic background. We fed two isogenic lines of rainbow trout (named A22h and N38h) with high carbohydrate diet (carbohydrate, 22.9%) or low carbohydrate diet (carbohydrate, 3.6%) for 12 weeks. Carbohydrates were associated with higher feed utilization owned by the well-known protein-sparing effect, with better fish growth performance. However, atypical regulation of glycolysis and gluconeogenesis in liver and absence of hk and glut4 induction in muscle, were also observed. Regarding the effects of carbohydrates on other metabolism, we observed an increased, at a molecular level, of hepatic cholesterol biosynthesis, fatty acid oxidation and mitochondrial energy metabolism. Genetic variability (revealed by the differences between the two isogenic lines) was observed for some metabolic genes especially for those involved in the EPA and DHA biosynthetic capacity. Finally, our study demonstrates that dietary carbohydrate not only affect glucose metabolism but also strongly impact the lipid and energy metabolism in liver and muscle of trout.

Composition of Intestinal Microbiota in Two Lines of Rainbow Trout (Oncorhynchus Mykiss) Divergently Selected for Muscle Fat Content.

BACKGROUND: Recently, studies suggest that gut microbiota contributes to the development of obesity in mammals. In rainbow trout, little is known about the role of intestinal microbiota in host physiology. OBJECTIVE: The aim of this study was to investigate the link between intestinal microbiota and adiposity, by high-throughput 16S RNA gene based illumina Miseq sequencing in two rainbow trout lines divergently selected for muscle lipid content. Fish from these two lines of rainbow trout are known to have a differing lipid metabolism. METHODS: Samples from the two lines (L for lean and F for fat) were collected from Midgut (M) and Hindgut (H) in juvenile fish (18 months) to compare intestinal microbiota diversity. RESULTS: Whatever the lines and intestinal localisation, Proteobacteria, Firmicutes and Actinobacteria are the dominant phyla in the bacterial community of rainbow trout (at least 97%). The results indicate that richness and diversity indexes as well as bacterial composition are comparable between all groups even though 6 specific OTUs were identified in the intestinal microbiota of fish from the fat line and 2 OTUs were specific to the microbiota of fish from the lean line. Our work contributes to a better understanding in microbial diversity in intestinal microbiota of rainbow trout. CONCLUSION: Altogether, our study indicates that no major modification of the intestinal microbiota is induced by selection for muscle lipid content and associated metabolic changes. Finally, we identified members of core microbiota in rainbow trout.

Origin of Public Memory B Cell Clones in Fish After Antiviral Vaccination.

Vaccination induces "public" antibody clonotypes common to all individuals of a species, that may mediate universal protection against pathogens. Only few studies tried to trace back the origin of these public B-cell clones. Here we used Illumina sequencing and computational modeling to unveil the mechanisms shaping the structure of the fish memory antibody response against an attenuated Viral Hemorrhagic Septicemia rhabdovirus. After vaccination, a persistent memory response with a public VH5JH5 IgM component was composed of dominant antibodies shared among all individuals. The rearrangement model showed that these public junctions occurred with high probability indicating that they were already favored before vaccination due to the recombination process, as shown in mammals. In addition, these clonotypes were in the naïve repertoire associated with larger similarity classes, composed of junctions differing only at one or two positions by amino acids with comparable properties. The model showed that this property was due to selective processes exerted between the recombination and the naive repertoire. Finally, our results showed that public clonotypes greatly expanded after vaccination displayed several VDJ junctions differing only by one or two amino acids with similar properties, highlighting a convergent response. The fish public memory antibody response to a virus is therefore shaped at three levels: by recombination biases, by selection acting on the formation of the pre-vaccination repertoire, and by convergent selection of functionally similar clonotypes during the response. We also show that naive repertoires of IgM and IgT have different structures and sharing between individuals, due to selection biases. In sum, our comparative approach identifies three conserved features of the antibody repertoire associated with public memory responses. These features were already present in the last common ancestors of fish and mammals, while other characteristics may represent species-specific solutions.

Detection of new pathways involved in the acceptance and the utilisation of a plant-based diet in isogenic lines of rainbow trout fry.

To meet the growing demand of fish feed for aquaculture, an increasing proportion of marine ingredients are being replaced by blends of plant products. However, the total replacement of marine ingredients in salmonid diets impairs fish performance. This is particularly true during the early fry stage and this stage is therefore considered of particular importance. In rainbow trout (RBT), the existence of a genetic variability to survive and grow with plant-based diets devoid of marine ingredients has now been proved, but the mechanisms behind are little studied especially at early stage. To investigate these, we analysed the whole transcriptome of three isogenic lines of RBT fry, which have similar growth when fed a marine resources-based diet (M diet) but which highly differ in their responses to a plant-based diet (V diet). Analysis of transcriptomes profiles revealed 1740, 1834 and 246 probes differentially expressed among the three genotypes when fed the V diet. The use of these lines led to the discovery of potential molecular markers linked to plant-based diet utilisation, some of them belonging to new pathways, never described before. An important number of genes was related to immunity, but further investigations are needed to better understand the difference between the genotypes in their immune status response to V diet exposure. Finally, differences in expression of genes related to feed intake and sensory perception among genotypes suggested that the mechanisms underlying the differences in growth on plant-based diet are closely linked to diet acceptance. Research on plants components affecting feed intake should be thus further explored.

Genetic and transcriptomic analyses provide new insights on the early antiviral response to VHSV in resistant and susceptible rainbow trout.

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is a major threat for salmonid farming and for wild fish populations worldwide. Previous studies have highlighted the importance of innate factors regulated by a major quantitative trait locus (QTL) for the natural resistance to waterborne VHSV infection in rainbow trout. The aim of this study was to analyze the early transcriptomic response to VHSV inoculation in cell lines derived from previously described resistant and susceptible homozygous isogenic lines of rainbow trout to obtain insights into the molecular mechanisms responsible for the resistance to the viral infection. RESULTS: We first confirmed the presence of the major QTL in a backcross involving a highly resistant fish isogenic line (B57) and a highly susceptible one (A22), and were able to define the confidence interval of the QTL and to identify its precise position. We extended the definition of the QTL since it controls not only resistance to waterborne infection but also the kinetics of mortality after intra-peritoneal injection. Deep sequencing of the transcriptome of B57 and A22 derived cell lines exposed to inactivated VHSV showed a stronger response to virus inoculation in the resistant background. In line with our previous observations, an early and strong induction of interferon and interferon-stimulated genes was correlated with the resistance to VHSV, highlighting the major role of innate immune factors in natural trout resistance to the virus. Interestingly, major factors of the antiviral innate immunity were much more expressed in naive B57 cells compared to naive A22 cells, which likely contributes to the ability of B57 to mount a fast antiviral response after viral infection. These observations were further extended by the identification of several innate immune-related genes localized close to the QTL area on the rainbow trout genome. CONCLUSIONS: Taken together, our results improve our knowledge in virus-host interactions in vertebrates and provide novel insights in the molecular mechanisms explaining the resistance to VHSV in rainbow trout. Our data also provide a collection of potential markers for resistance and susceptibility of rainbow trout to VHSV infection.

Hepatic glucose metabolic responses to digestible dietary carbohydrates in two isogenic lines of rainbow trout.

Rainbow trout (Oncorhynchus mykiss) was recognized as a typical 'glucose-intolerant' fish and poor dietary carbohydrate user. Our first objective was to test the effect of dietary carbohydrates themselves (without modification of dietary protein intake) on hepatic glucose gene expression (taking into account the paralogs). The second aim was to research if two isogenic trout lines had different responses to carbohydrate intake, showing one with a better use dietary carbohydrates. Thus, we used two isogenic lines of rainbow trout (named A32h and AB1h) fed with either a high carbohydrate diet or a low carbohydrate diet for 12 weeks. We analysed the zootechnical parameters, the plasma metabolites, the hepatic glucose metabolism at the molecular level and the hormonal-nutrient sensing pathway. Globally, dietary carbohydrate intake was associated with hyperglycaemia and down regulation of the energy sensor Ampk, but also with atypical regulation of glycolysis and gluconeogenesis in the liver. Indeed, the first steps of glycolysis and gluconeogenesis catalysed by the glucokinase and the phospenolpyruvate carboxykinase are regulated at the molecular level by dietary carbohydrates as expected (i.e. induction of the glycolytic gck and repression of the gluconeogenic pck); by contrast, and surprisingly, for two other key glycolytic enzymes (phosphofructokinase enzyme - pfkl and pyruvate kinase - pk) some of the paralogs (pfklb and pklr) are inhibited by carbohydrates whereas some of the genes coding gluconeogenic enzymes (the glucose-6-phosphatase enzyme g6pcb1b and g6pcb2a gene and the fructose1-6 biphosphatase paralog fbp1a) are induced. On the other hand, some differences for the zootechnical parameters and metabolic genes were also found between the two isogenic lines, confirming the existence of genetic polymorphisms for nutritional regulation of intermediary metabolism in rainbow trout. In conclusion, our study determines some new and unexpected molecular regulations of the glucose metabolism in rainbow trout which may partly lead to the poor utilization of dietary carbohydrates and it underlines the existence of differences in molecular regulation of glucose metabolism between two isogenic lines which provides arguments for future selection of rainbow trout.

Regulation of genes related to cholesterol metabolism in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet.

When compared with fish meal and fish oil, plant ingredients differ not only in their protein content and amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. To investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1 g body wt for 6 mo with a totally plant-based diet (V), a marine diet (M), and a marine-restricted diet (MR), with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis, and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were upregulated in trout fed the V diet, whereas expression of genes related to bile acid synthesis ( cyp7a1) and cholesterol elimination ( abcg8) were reduced. Feeding trout the V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα, and miR-223. However, plasma and body cholesterol levels in trout fed the V diet were lower than in fish fed the M diet, raising the question of the role of cholesterol in the negative effect of plant-based diet on growth.