Evolution and Expression of the Immune System of a Facultatively Anadromous Salmonid.

Vertebrates have evolved a complex immune system required for the identification of and coordinated response to harmful pathogens. Migratory species spend periods of their life-cycle in more than one environment, and their immune system consequently faces a greater diversity of pathogens residing in different environments. In facultatively anadromous salmonids, individuals may spend parts of their life-cycle in freshwater and marine environments. For species such as the brown trout Salmo trutta, sexes differ in their life-histories with females more likely to migrate to sea while males are more likely to stay and complete their life-cycle in their natal river. Salmonids have also undergone a lineage-specific whole genome duplication event, which may provide novel immune innovations but our current understanding of the differences in salmonid immune expression between the sexes is limited. We characterized the brown trout immune gene repertoire, identifying a number of canonical immune genes in non-salmonid teleosts to be duplicated in S. trutta, with genes involved in innate and adaptive immunity. Through genome-wide transcriptional profiling ("RNA-seq") of male and female livers to investigate sex differences in gene expression amplitude and alternative splicing, we identified immune genes as being generally male-biased in expression. Our study provides important insights into the evolutionary consequences of whole genome duplication events on the salmonid immune gene repertoire and how the sexes differ in constitutive immune expression.

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.

Immunomodulatory function of antimicrobial peptide EC-Hepcidin1 modulates the induction of inflammatory gene expression in primary cells of Caspian Trout (Salmo trutta caspius Kessler, 1877).

Hepcidins, a group of antimicrobial peptides (AMPs), play a key role in the innate immune system of fishes and act against different pathogens. In this study, antimicrobial and immune-inflammatory activity of a synthetic EC-hepcidin1, previously identified from orange-spotted grouper, were evaluated. EC-hepcidin1 showed weak activity against the zoonotic fish pathogen Streptococcus iniae (MIC 100 µg mL-1 and MBC 150 µg mL-1). To study the effect of AMPs in general, and EC-hepcidin1 in particular, a primary cell culture (SC) from the fin tissue of the Caspian Trout (Salmo trutta caspius) was established. The neutral Red method on SC cells revealed that EC-hepcidin1 has no or very low cytotoxic properties. Treatment of cells with either EC-hepcidin1 (150 µg mL-1) or fish pathogen Streptococcus iniae (MOI = 10) and a mixture of both resulted in the up-regulation of gene expression of MHC-UBA, IL-6, and TNFα indicating the modulatory function on inflammatory processes. These findings indicate that EC-hepcidin1 might act as a candidate for modulation of the innate immune system in S. iniae-based infection.

Acute ammonia poisoning in dolly varden char (Salvelinus malma) and effect of methionine sulfoximine.

Ammonia is toxic to most bony fishes. However, little information is available on the toxicology mechanisms induced by ammonia and the means to mitigate the effects by various fishes. In this study, four groups of experiments were designed and carried out to test the response of dolly varden char to ammonia toxicity and their mitigation through methionine sulfoximine (MSO). NaCl group was injected with NaCl, NH3 group was injected with ammonium acetate, NH3+MSO group was injected with ammonium acetate and MSO, MSO group was injected with MSO. Results showed that ammonia toxicity could lead to blood deterioration (elevation in white blood cell and blood ammonia), free amino acid imbalance (elevation in glutamine, glutamate, arginine and ornithine, coupled with reduction of citrulline and aspartate), ammonia metabolism enzyme activity inhibition (reduction in carbamyl phosphate synthetase, ornithine transcarbamylase and arginase), oxidative stress (reduction in superoxide dismutase, catalase and glutathione peroxidase) and immunosuppression (reduction in lysozyme, 50% hemolytic complement, total immunoglobulin and phagocytic index), but the MSO can eliminate fatal effect of oxidative damage. In addition, ammonia poisoning could induce down-regulation of antioxidant enzymes coding genes (SOD, CAT and GPx) and up-regulation of inflammatory cytokine genes (TNFα, IL-1ß and IL-8) transcription, suggesting that immunosuppression and inflammation may relate to oxidative stress in fish.

Identification and molecular characterization of CD4 genes in brown trout (Salmo trutta).

CD4+ cells are vital in coordinating the immune response against pathogens. In the present study, three different CD4 homologs, namely, CD4-1, CD4-2a, and CD4-2b were identified and characterized. Further, their basal expression levels in different brown trout (Salmo trutta) tissues were also investigated. CD4-1 was 1473 nucleotides long, with an open reading frame (ORF) encoding 490 amino acids with four immunoglobulin superfamily-like domains. CD4-2a and CD4-2b like genes were 945 and 999 nucleotides long containing ORFs with 313 and 331 amino acids, respectively. The brown trout CD4-1 protein sequence demonstrated a 95% and 89% identity with Atlantic salmon and rainbow trout CD4-1 genes, respectively. On the other hand, brown trout CD4-2a and CD4-2b protein sequences presented an identity of 84% and 97.7% with rainbow trout and Atlantic salmon, respectively. The basal expression levels of the identified brown trout CD4-genes were investigated, which were higher in thymus, spleen, and head kidney than in those the gills, liver, intestine, heart, and brain tissues.

Immunoassays are not immune to errors: Examples from two studies of steroid output from freshwater trout farms.

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L-1) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L-1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed.

Transcriptome profiling of posterior kidney of brown trout, Salmo trutta, during proliferative kidney disease.

BACKGROUND: Tetracapsuloides bryosalmonae is a myxozoan parasite which causes economically important and emerging proliferative kidney disease (PKD) in salmonids. Brown trout, Salmo trutta is a native fish species of Europe, which acts as asymptomatic carriers for T. bryosalmonae. There is only limited information on the molecular mechanism involved in the kidney of brown trout during T. bryosalmonae development. We employed RNA sequencing (RNA-seq) to investigate the global transcriptome changes in the posterior kidney of brown trout during T. bryosalmonae development. METHODS: Brown trout were exposed to the spores of T. bryosalmonae and posterior kidneys were collected from both exposed and unexposed control fish. cDNA libraries were prepared from the posterior kidney and sequenced. Bioinformatics analysis was performed using standard pipeline of quality control, reference mapping, differential expression analysis, gene ontology, and pathway analysis. Quantitative real time PCR was performed to validate the transcriptional regulation of differentially expressed genes, and their correlation with RNA-seq data was statistically analyzed. RESULTS: Transcriptome analysis identified 1169 differentially expressed genes in the posterior kidney of brown trout, out of which 864 genes (74%) were upregulated and 305 genes (26%) were downregulated. The upregulated genes were associated with the regulation of immune system process, vesicle-mediated transport, leucocyte activation, and transport, whereas the downregulated genes were associated with endopeptidase regulatory activity, phosphatidylcholine biosynthetic process, connective tissue development, and collagen catabolic process. CONCLUSION: To our knowledge, this is the first RNA-seq based transcriptome study performed in the posterior kidney of brown trout during active T. bryosalmonae development. Most of the upregulated genes were associated with the immune system process, whereas the downregulated genes were associated with other metabolic functions. The findings of this study provide insights on the immune responses mounted by the brown trout on the developing parasite, and the host molecular machineries modulated by the parasite for its successful multiplication and release.

Overcoming Fish Defences: The Virulence Factors of Yersinia ruckeri.

Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

Effects of dietary b-glucan, mannan oligosaccharide, Lactobacillus plantarum and their combinations on growth performance, immunity and immune related gene expression of Caspian trout, Salmo trutta caspius (Kessler, 1877).

This study investigated the effect of individual and combination of dietary pre- and probiotics (ß-glucan, 3 mg/g; mannan oligosaccharide (MOS), 4 mg/g; and Lactobacillus plantarum; 108 CFU/mg diet) on growth performance, blood immune parameters, expression of immune related genes, and intestinal microbial of Caspian trout (Salmo trutta caspius). On the basis of feeding with immunostimulant diets, the fish were assigned into eight groups denoted as: control (basal diet), bß (basal diet + ß-glucan), bM (basal diet + MOS), bLp (basal diet + L. plantarum), bßLp (basal diet + ß-glucan + L. plantarum), bMLp (basal diet + MOS + L. plantarum), bMß (basal diet + MOS + ß-glucan), and bMßLp (basal diet + MOS + ß-glucan + L. plantarum). All of the immunostimulant diets, in general, reduced feed intake (FI) and food conversion ratio (FCR) and increased WG, PER, and final weight. Condition factor (CF) demonstrated the lowest level in the experimental group received bMßLp. Total lipid increased in the fish received the additives, especially bM and bMß. Ash content demonstrated significant increase in the fish fed on bß and bMßLp, whereas moisture content was reduced in the group fed with L. plantarum-supplemented diet. All immunostimulant diets enhanced the activity and levels of lysozyme, Immunoglobulin M (IgM), and serum alternative complement activity (ACH50); the highest value for these indices was observed in the groups fed with bMß, bMßLp, and bßLp. bMß-treated fish group displayed the highest cortisol and glucose levels. bM diet induced the highest mRNA transcription of TNF-α1 in head kidney, whereas bLp, bMß, and bMßLp showed no effect. IL1ß exhibited the greatest up-regulation, about 8.75 fold change, in response to the diet supplemented only with ß-glucan. bßLp and bß significantly enhanced the relative IL-8 mRNA expression in the head kidney (about 2.75 and 1.9 folds, respectively), yet in response to bMßLp treatment it showed a decrease of about 5.7 times lower than the control group. In addition, intestinal population of L. plantarum showed the highest loads in the groups fed on the diets which were treated with the probiotic. Taken together, combinational use of these immunostimulants enhanced humoral innate immune system, whereas their individual and combinational application could increase and decrease the transcription of inflammation-related genes, respectively.

Effect of dietary soybean lecithin on fish performance, hemato-immunological parameters, lipid biochemistry, antioxidant status, digestive enzymes activity and intestinal histomorphometry of pre-spawning Caspian brown trout (Salmo trutta caspius).

A feeding experiment was conducted to evaluate the effects of dietary soybean lecithin (SBL) supplementation on performance, hemato-immunological parameters, lipid biochemistry, antioxidant status, digestive enzymes activity and intestinal histomorphometry of Caspian brown trout, Salmo trutta caspius in the pre-spawning stage. The basal diet was supplemented with 0% (control), 3%, 6%, 9% and 12% of SBL to obtain five experimental diets. Fish with an average weight of 350 ±â€¯10 g were randomly distributed among five experimental groups and fed for 90 days. Dietary SBL resulted in better performance including specific growth rate (SGR), weight gain (WG) and feed conversion ratio (FCR) (p < 0.05). Among the different hemato-immunological parameters, white blood cell counts (WBC), lysozyme, alternative complement activity (ACH50) and total immunoglobulin (IgM) content of serum were significantly increased with dietary SBL inclusion (p < 0.05). For antioxidant enzymes, glutathione S-transferase (GST) and catalase (CAT) showed significant differences among various experimental diets (p < 0.05). Furthermore, digestive enzymes activity including alkaline protease, lipase and amylase were increased in those fish received SBL supplemented diets (p < 0.05). Our results revealed that the dietary SBL improved some physiological responses of the fish and indicate 6-9% dietary SBL supplementation would improve the physiological competence of the pre-spawning Caspian brown trout breeders.

Cloning, characterization and tissue-specific expression of the antimicrobial peptide hepcidin from caspian trout (Salmo caspius) and the antibacterial activity of the synthetic peptide.

Antimicrobial peptides have a wide range of antimicrobial activity and widely occur in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine-rich antimicrobial peptides that are active against a variety of pathogens including gram-positive and gram-negative bacteria, as well as viruses. In this study, the hepcidin gene of Caspian trout (CtHep) was identified and characterized. Our results showed that CtHep cDNA has a 267-bp Open Reading Frame (ORF), which is translated to 88 amino acids. The CtHep was classified in the HAMP1 class of hepcidins. Comparison of DNA and cDNA sequences showed that CtHep has 3 exons and 2 introns. The signal, prodomain and mature part of CtHep have 24, 39 and 25 amino acids, respectively. The mature peptide has a molecular weight of 2881.43 Da and a theoretical isoelectric point of 8.53. The expression of CtHep mRNA was detected in different tissues of healthy and infected fish. CtHep expression in the liver, head kidney, spleen and skin was significantly enhanced after bacterial challenge. Expression of CtHep in different embryonic development stages was also substantial. Antibacterial activity of synthetic CtHep peptides was investigated against a number of Gram-positive and Gram-negative bacteria. CtHep inhibited some pathogenic bacteria such as Streptococcus iniae and Aeromonas hydrophila. In the in vivo experiment, CtHep upregulated the cytokines IL-6 and TNF-α in both kidney and spleen tissues after 24 h of the peptide injection. In conclusion, our study showed that CtHep plays an important role in the immune system of Caspian trout and also in the embryonic stages. Moreover, CtHep peptide has a potential to be used as an antimicrobial therapeutic agent as well as an immunostimulant in aquaculture.

Comparative susceptibilities and immune-related gene expressions of brown trout strains and their hybrids infected with Lactococcus garvieae and Yersinia ruckeri.

Brown trout are polymorphic salmonid species, and it is of importance to investigate whether hybridization affects disease resistance. In this study, susceptibility of brown trout (Salmo trutta Abant, Anatolian, Black Sea, and Caspius) strains and their hybrids to Lactococcus garvieae and Yersinia ruckeri as well as their immune-related gene expression profiles were studied. Results indicated that reciprocal hybridization did not affect disease resistance in brown trout strains. Purebred Black Sea strain of brown trout was the most resistant group against Y. ruckeri, followed by other Black Sea strain hybrids. On the other hand, purebred Anatolian strain was the most resistant group to L. garvieae, followed by other Anatolian strain hybrids. Expression pattern of target genes differed in families, but the overall gene expression was comparatively high in Y. ruckeri infected families. Upregulations were mainly significant at 7 and 28 d post infection while marginal regulations were observed 8 h after infection. Disease resistance status of strains was supported by high expression of immune-related genes such as major histocompatibility complex class I (MHC-I), immunoglobulin light chain (IgL), and antioxidant- and hemoglobin-related gene expression. Therefore, our findings suggest that Black Sea and Anatolian strains could be used to develop fish stock that are resistant for yersiniosis and lactocaccosis, respectively.

Vaccine-Induced Protection Against Furunculosis Involves Pre-emptive Priming of Humoral Immunity in Arctic Charr.

With respect to salmonid aquaculture, one of the most important bacterial pathogens due to high mortality and antibiotic usage is the causative agent of typical furunculosis, Aeromonas salmonicida spp. salmonicida (Asal). In Atlantic salmon, Salmo salar, the host response during infections with Asal is well-documented, with furunculosis outbreaks resulting in significant mortality in commercial settings. However, less is known about the host-pathogen interactions in the emerging aquaculture species, Arctic charr Salvelinus alpinus. Furthermore, there is no data on the efficacy or response of this species after vaccination with commonly administered vaccines against furunculosis. To this end, we examined the immunological response of S. alpinus during infection with Asal, with or without administration of vaccines (Forte Micro®, Forte Micro® + Renogen®, Elanco Animal Health). Artic charr (vaccinated or unvaccinated) were i.p.-injected with a virulent strain of Asal (106 CFUs/mL) and tissues were collected pre-infection/post-vaccination, 8, and 29 days post-infection. Unvaccinated Arctic charr were susceptible to Asal with 72% mortalities observed after 31 days. However, there was 72-82% protection in fish vaccinated with either the single or dual-vaccine, respectively. Protection in vaccinated fish was concordant with significantly higher serum IgM concentrations, and following RNA sequencing and transcriptome assembly, differential expression analysis revealed several patterns and pathways associated with the improved survival of vaccinated fish. Most striking was the dramatically higher basal expression of complement/coagulation factors, acute phase-proteins, and iron hemostasis proteins in pre-challenged, vaccinated fish. Remarkably, following Asal infection, this response was abrogated and instead the transcriptome was characterized by a lack of immune-stimulation compared to that of unvaccinated fish. Furthermore, where pathways of actin assembly and FcγR-mediated phagocytosis were significantly differentially regulated in unvaccinated fish, vaccinated fish showed either the opposite regulation (ForteMicro®), or no impact at all (ForteMicro®Renogen®). The present data indicates that vaccine-induced protection against Asal relies on the pre-activation and immediate control of humoral immune parameters that is coincident with reduced activation of apoptotic (e.g., NF-κB) and actin-associated pathways.

Identification and expression of the hepcidin gene from brown trout (Salmo trutta) and functional analysis of its synthetic peptide.

Hepcidin, a hepatic antimicrobial peptide, is a key player of the nonspecific immune system. The structure of hepcidin gene from brown trout (Bthepc) has been characterized at the molecular level. The 1158-bp mRNA generates a coding sequence (CDS) of 267 bp, which encodes an 88-amino acid protein. Molecular evolution analysis classified Bthepc to the family Salmonidae. Amino acid sequence homologies between Bthepc and hepcidin in other species such as Oncorhynchus mykiss, Salmo salar, and Hucho taimen were found to be 93.18%, 96.59%, and 92.05% respectively. The mature peptide and the signal peptide of Bthepc are made of 25 and 24 amino acids, respectively. Similar to the other species, eight conserved cysteines in the mature peptide of Bthepc are held together by four disulphide bonds. Expression profiling of Bthepc indicated its highest expression in the liver. Further, iron levels or inflammation did not induce the age-dependent expression of Bthepc. Bthepc mRNA expression analysis in six immune tissues (liver, gill, spleen, skin, head kidney and intestine) indicated different levels of increase when challenged with Aeromonas salmonicida and Aeromonas hydrophila. The antimicrobial activity of synthetic Bthepc to typical pathogens was verified in vitro. In addition, Bthepc showed moderate haemolytic activity to mammalian erythrocytes. The antimicrobial activity of Bthepc was attributed to the disruption of the bacterial outer membrane integrity, which was evident from our scanning electron microscopy results. In summary, hepcidin gene of brown trout was characterized, and its antimicrobial activity was verified on different levels.

Do the toll-like receptors and complement systems play equally important roles in freshwater adapted Dolly Varden char (Salvelinus malma)?

Unlike the normal anadromous lifestyle, Chinese native Dolly Varden char (Salvelinus malma) is locked in land and lives in fresh water lifetime. To explore the effect of freshwater adaption on its immune system, we constructed a pooled cDNA library of hepatopancreas and spleen of Chinese freshwater Dolly Varden char (S. malma). A total of 27,829 unigenes were generated from 31,233 high-quality transcripts and 17,670 complete open reading frames (ORF) were identified. Totally 25,809 unigenes were successfully annotated and it classified more native than adaptive immunity-associated genes, and more genes involved in toll-like receptor signal pathway than those in complement and coagulation cascades (51 vs 3), implying the relative more important role of toll-like receptors than the complement system under bacterial injection for the freshwater Dolly Varden char. These huge different numbers of TLR and complement system identified in freshwater Dolly Varden char probably caused by distinct evolution pressure patterns between fish TLR and complement system, representative by TLR3 and TLR5 as well as C4 and C6, respectively, which were under purifying and positively selecting pressure, respectively. Further seawater adaptation experiment and the comparison study with our library will no doubt be helpful to elucidate the effect of freshwater adaption of Chinese native Dolly Varden char on its immune system.

The Effects of a Synbiotic Mixture of Galacto-Oligosaccharides and Bacillus Strains in Caspian Salmon, Salmo trutta caspius Fingerlings.

The effect of dietary supplementation with a synbiotic mixture of galacto-oligosaccharides (GOS) and Bacillus spp. was examined in Caspian salmon, Salmo trutta caspius (Kessler, 1877) fingerlings. Caspian salmon fed with the synbiotic diet had significantly higher weight gain rate, protein efficiency ratio, and survival rate, as well as lower feed conversion ratio, compared to the control group (P < 0.05). The serum protein, albumin, globulin, and lactate dehydrogenase levels of the fish fed with the synbiotic diet were significantly higher than the control group (P < 0.05), while the serum alkaline phosphatase levels were significantly lower (P < 0.05). The activities of the innate immune response parameters, including lysozyme, superoxide dismutase, and catalase were significantly higher in the Caspian salmon fed with the synbiotic diet (P < 0.05). The gut microbiota of the Caspian salmon fed with the synbiotic diet contained significantly elevated total viable aerobic bacterial counts (TVABCs), lactic acid bacteria (LAB) levels, and LAB/TVABCs ratio (P < 0.05). Additionally, the gut activities of amylase, trypsin, and chymotrypsin in the gut, as well as the trypsin/chymotrypsin ratio, were significantly increased in the fish that received the synbiotic diet (P < 0.05). In conclusion, the combined GOS and Bacillus spp. supplement positively affected the growth, survival rate, immunobiochemical parameters, digestive activity, and beneficial microbial density in the gut of Caspian salmon fingerlings.

Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

Small-scale intraspecific patterns of adaptive immunogenetic polymorphisms and neutral variation in Lake Superior lake trout.

Many fishes express high levels of intraspecific variability, often linked to resource partitioning. Several studies show that a species' evolutionary trajectory of adaptive divergence can undergo reversals caused by changes in its environment. Such a reversal in neutral genetic and morphological variation among lake trout Salvelinus namaycush ecomorphs appears to be underway in Lake Superior. However, a water depth gradient in neutral genetic divergence was found to be associated with intraspecific diversity in the lake. To investigate patterns of adaptive immunogenetic variation among lake trout ecomorphs, we used Illumina high-throughput sequencing. The population's genetic structure of the major histocompatibility complex (MHC Class IIß exon 2) and 18 microsatellite loci were compared to disentangle neutral and selective processes at a small geographic scale. Both MHC and microsatellite variation were partitioned more by water depth stratum than by ecomorph. Several metrics showed strong clustering by water depth in MHC alleles, but not microsatellites. We report a 75% increase in the number of MHC alleles shared between the predominant shallow and deep water ecomorphs since a previous lake trout MHC study at the same locale (c. 1990s data). This result is consistent with the reverse speciation hypothesis, although adaptive MHC polymorphisms persist along an ecological gradient. Finally, results suggested that the lake trout have multiple copies of the MHC II locus consistent with a historic genomic duplication event. Our findings indicated that conservation approaches for this species could focus on managing various ecological habitats by depth, in addition to regulating the fisheries specific to ecomorphs.

Oral DNA vaccines based on CS-TPP nanoparticles and alginate microparticles confer high protection against infectious pancreatic necrosis virus (IPNV) infection in trout.

Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a contagious viral disease causing remarkable mortalities in different fish species. Despite the availability of commercial vaccines against IPN, the disease still constitutes one of the main threats to the aquaculture industry worldwide. In this study, we developed a DNA vaccine encoding the VP2 gene of IPNV and evaluated its ability to induce protective immunity in rainbow trout fry (3 g) at doses of 10 and 25 µg/fish and boosting with the same doses two weeks later through the oral route using chitosan/tripolyphosphate (CS-TPP) nanoparticles and alginate microparticles incorporated into fish feed. The distribution of the administered vaccines in different organs and transcription of VP2 gene were confirmed by RT-PCR assay at day 30 post boost-vaccination. Transcript levels of IFN-1, Mx-1, IgM, IgT and CD4 genes was dependent on vaccine dose and was significantly up-regulated in head kidney of all orally vaccinated fish groups compared to controls (pcDNA3.1). Cumulative mortalities post-challenge with virulent isolate of the virus were lower in the vaccinated fish and a relative percentage survival (RPS) of 59% and 82% were obtained for the 10 and 25 µg/fish pcDNA3.1-VP2 groups, respectively. Vaccination with the same amount of pcDNA3.1-VP2 encapsulated with CS-TPP nanoparticles resulted in RPS of 47 %and 70%, respectively. Detectable anti-IPNV antibodies were shown until 90 days postvaccination. The orally administrated vaccines significantly decreased VP4 transcripts thus contributing to reducing viral load in surviving fish on day 45 post-challenge. In conclusion, these results show good to high protection post-vaccination alongside with significant up-regulation of key immune genes and detectable levels of circulating antibodies after oral administration of the DNA vaccine formulated in CS-TPP nanoparticles and alginate microparticles in fish feed.

Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated ß-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.