This study addressed the escalating demand for aquatic feed by exploring the potential of alfalfa nutrient concentrate (ANC) as feed ingredient for rainbow trout. Test diets contained varying ANC levels (0%, 5%, 10%, 15%, and 20%) to replace fishmeal (32% in the 0% ANC diet) to achieve equal digestible protein and were processed using a cooking extrusion method. Analysis of feed pellets showed that pellet density increased with ANC levels (P < 0.001), resulting in sinking pellets at 20% ANC. Water stability and durability were improved while oil leakage decreased with increasing ANC levels (P < 0.05). Two feeding trials were conducted to test the diets in flow-through water systems with three replicates per diet. The first 10-week trial evaluated their impact on feeding, fecal physical quality, and the apparent digestibility coefficient (ADC) of dietary nutrients in rainbow trout (initial body weight 18.0 ± 0.2 g). ANC inclusion did not impact the palatability and satiety feed intake of the fish (P > 0.05). However, the ADC of dry matter and phosphorus significantly decreased in fish fed the 20% ANC diet (P < 0.05). The second 9-week trial investigated the growth performance, nutrition quality, and metabolism of rainbow trout (initial body weight 19.0 ± 0.2 g). While all fish exhibited substantial growth, fish fed diets with 10% to 20% ANC displayed lower specific growth rate and higher feed conversion ratio compared to those fed with 0% or 5% ANC (P < 0.05). The whole body protein content was higher in fish fed 5% ANC compared to all other treatments (P = 0.030). The biochemical parameters of plasma were similar across treatments, except for a decrease in plasma phosphorus levels in fish fed a 10% ANC diet compared to those fed a 0% ANC diet (P = 0.033). Significant changes were observed in liver metabolism including tricarboxylic acid cycle, amino acid and energy metabolism pathways in fish fed the 20% ANC diet versus the 0% ANC diet (P < 0.05). These results demonstrate that ANC inclusion improved pellet physical quality without impairing feeding behavior and nutritional quality of the fish but inclusion ≥10% in the diet reduced fish growth. This study offers the first comprehensive assessment of the potential of ANC used in fish feed involving feed management, feeding evaluation, and the biological response.

Ghrelin is a peptide hormone/cytokine that regulates metabolic processes and plays essential roles in the immune system. To evaluate the immunomodulatory actions of ghrelin isoforms in rainbow trout (RT), an in vitro model was utilized with primary cells isolated from fish head kidney (HKD). These RT-HKD cells were treated with synthetic rainbow trout ghrelin and its truncated isoform, desVRQ-ghrelin, over time (0, 2, 4, and 24 h). Reverse transcriptase-coupled qPCR was used to measure the differential expression patterns of genes relevant to various immune processes and genes of antimicrobial peptides. Ghrelin isoform treatments resulted in functional perturbations that displayed overlapping and divergent patterns of gene expression. The differing actions between the two ghrelin isoforms on various assessed genes, and at differing time points, suggested that the two analogs may activate unique pathways, thereby eliciting distinct responses in fish immunity.

Hepcidin antimicrobial peptide (hamp) is active in teleosts against invading pathogens and plays important roles in the stress and immune responses of finfish. The response of hamp gene was studied in yellow perch (yp) (Perca flavescens) challenged with lipopolysaccharides to understand if this immunity response is sex-specifically different. The cloned hamp gene consists of an open-reading frame of 273 bp and encodes a deduced protein of 90 amino acids (a.a.), which includes a signal peptide of 24 a.a., a pro-domain of 40 a.a. and a mature peptide of 26 a.a. Yp hamp involves 8 cysteine residues with 4 disulfide bonds, and a protein with an internal alpha helix flanked with C- and N-terminal random coils was modeling predicted. RT-qPCR was used to analyze the relative abundances (RAs) of hamp mRNA in the livers of juvenile female and male yellow perch challenged with lipopolysaccharide. The expression levels of hamp were significantly elevated by 3 h (RA = 7.3) and then peaked by 6 h (RA = 29.4) post-treatment in females but the peak was delayed to 12 h (RA = 65.4) post-treatment in males. The peak mRNA level of challenged males was shown 7.6-fold higher than females. The post-treatment responses in both genders decreased to their lowest levels by 24 h and 48 h. Overall, female perch had an earlier but less-sensitive response to the lipopolysaccharide challenge than male.

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

Microplastics are emergent contaminants threatening aquatic organisms including aquacultured fish. This study investigated the effects of high-density polyethylene (HDPE, 100 to 125 μm) on yellow perch (Perca flavescens) based on integrative evaluation including growth performance, nutritional status, nutrient metabolism, fish health, and gut microbial community. Five test diets (0, 1, 2, 4, or 8 g HDPE/100 g diet) containing 41% protein and 10.5% lipid were fed to juvenile perch (average body weight, 25.9 ± 0.2 g; n = 15) at a feeding rate of 1.5% to 2.0% body weight daily. The feeding trial was conducted in a flow-through water system for 9 wk with 3 tanks per treatment and 15 yellow perch per tank. No mortality or HDPE accumulation in the fish was found in any treatments. Weight gain and condition factor of fish were not significantly impacted by HDPE (P > 0.05). Compared to the control group, fish fed the 8% HDPE diet had significantly decreased levels of protein and ash (P < 0.05). In response to the increasing levels of HDPE exposure, the hepatosomatic index value, hepatocyte size, and liver glycogen level were increased, but lipid content was reduced in the liver tissues. Compared to the control treatment, fish fed the 8% HDPE diet had significant accumulations of total bile acids and different metabolism pathways such as bile acid biosynthesis, pyruvate metabolism, and carnitine synthesis. Significant enterocyte necrosis was documented in the foregut of fish fed the 2% or 8% HDPE diet; and significant cell sloughing was observed in the midgut and hindgut of fish fed the 8% HDPE diet. Fish fed the 2% HDPE diet harbored different microbiota communities compared to the control fish. This study demonstrates that HDPE ranging from 100 to 125 μm in feed can be evacuated by yellow perch with no impact on growth. However, dietary exposure to HDPE decreased whole fish nutrition quality, altered nutrient metabolism and the intestinal histopathology as well as microbiota community of yellow perch. The results indicate that extended exposure may pose a risk to fish health and jeopardize the nutrition quality of aquacultured end product. This hypothesis remains to be investigated further.

Abstract Background Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and are thusly notifiable diseases with the World Organization for Animal Health. This study assessed the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores the abilities of specific IHNV genes to interfere with the interferon pathway in heterogenous teleost cell-lines. Methods Optimized protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (-IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 hours post transfection (hpt) with luciferase constructs driven by a modified β-Actin promoter (pCAG). Their modulation of the innate antiviral immune response was characterized 72 hpt, using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation, upon MAVS co-transfection. Results M was generally confirmed as the strongest constitutive transcriptional suppressor while IHNV P, but not VHSV P, augmented constitutive transcription in fibroblastic cell types. Cell-specific effects were observed for viral G gene, with VHSV G exhibiting suppression of basal transcription in EPC and BF-2 but not in trout cells; while IHNV G was stimulatory in RTG-2, but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV NV compared to VHSV NV. The innate antiviral immune response, focusing on the IFN pathway, was silenced by IHNV M in all cell lines tested. IHNV N showed a dose-dependent suppression of type I IFN, but with minor effects on MX-1. IHNV P and G played minor IFN-inhibitory roles, consistent and dose-dependent only for G in rainbow trout cells. IHNV NV mediated a consistent stimulatory effect on either Type I IFN or MX-1, but much less pronounced in RTgill-W1. Conclusions This study extends our understanding of Novirhabdoviruses-host interaction, showing differential innate immune responses in heterogenous cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128-133 aa in length, and mature peptides of 109-111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2-3 fold) of five (nkl 2-4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (similar to 57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.

This study determined whether accumulated liver and visceral lipid in yellow perch can be alleviated by feeding an appropriate starch source. We compared the growth performance, nutritional composition and health of yellow perch fed a control diet (no added starch) and three diets with corn, potato or wheat starch (200 g/kg diet) to replace an equal proportion of fishmeal in the control diet. Dietary starch led to increased bulk density, hardness and gelatinization levels of feed pellets compared with the control diet. At the end of an 8-week feeding, all fish fed the test diets had similar growth rates (p > .05). However, the wheat starch diet resulted in significantly enlarged livers and increased accumulation of lipid in the liver, viscera and whole body (p < .05). This diet also caused significantly higher levels of fasting glucose, aspartate aminotransferase and alkaline phosphatase activities in serum than the control diet. Liver cell diameters were larger, and Kupffer cell numbers were lower in the fish fed the wheat starch than those fed the control diet. Potato or corn starch had less impact on fish health and is considered to be an appropriate carbohydrate sources for yellow perch under the current testing conditions.

Yellow perch (Perca favescens) is an important native North American fish in ecology and aquaculture. However, molecular markers are minimal for this species, especially those with genome-wide distribution. Single sequence repeat markers are always developed based on sequences from a single individual, which is time consuming with a low success rate. In this study, combining data from HiSeq and MiSeq platforms, we applied restriction site-associated DNA sequencing (RAD-Seq) to yellow perch geographic demes to develop large numbers of polymorphic genetic markers, including single nucleotide polymorphisms and simple sequence repeats, and to evaluate nucleotide diversity. A total of 258,056 contigs were assembled with the length of 605 ± 71 (mean ± SD) bp, which were longer than those just using HiSeq data. Nucleotide diversity (π) was 0.00304 with 95% confidence intervals from 0.00303 to 0.00304. Based on the assembled sequences, a total of 59,766 perfect single sequence repeats were detected, with AC/GT being the most common repeat motif and GC being the rarest. Based on this breeding population, 41,736 high-quality single nucleotide polymorphisms and 10,412 polymorphic single sequence repeats were obtained. Validation of amplification by randomly selecting 40 polymorphic single sequence repeats showed a high success rate (85%). Our approach for polymorphic single sequence repeats development enables new usage of data from RAD-seq techniques. These developed markers could be used in finfish broodstock management, genetic improvement and resource conservation, and would contribute to sustainable development of fisheries and the aquaculture industries.

Two 3-week growth trials were performed to determine the optimum feeding rate of juvenile lake sturgeon (Acipenser fulvescens) fed with a semi-purified dry diet (protein, 440 g; lipid, 130 g/kg) at different feeding rates: 5, 10, 20, 30, and 40 g/kg body weight per day (BW/.d-1), for 36 g-sized fish; and 2.5, 5, 10, 20, and 30 g/kg BW/.d-1, for 80 g sized fish. Feeding rates significantly influenced growth performances, feed conversion ratio, wholebody lipid and moisture contents, energy and protein retention, and fasted blood glucose levels (p < 0.05). The ratio of total C/total N measured in whole fish was positively correlated to the feeding rates and the lipid contents of whole fish. Suboptimal feeding significantly depressed growth performance and protein synthesis based on the measurement of RNA/DNA ratio in white muscle. Based on polynomial regression models, an optimal feeding rate to support the maximum growth, protein and energy retention was 34.5, 22 and 27.3 g/kg BW/.d-1, respectively, for fish sized at 36-72 g; and 29.0, 16.9 and 19.9 g/kg BW/.d-1, respectively for fish sized at 80-140 g. Results of this study will benefit future research to develop practical feeds to replace frozen live feed and feed management for lake sturgeon.

Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1β: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1β mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1β, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout.

Ghrelin, a gut-brain peptide hormone, is implicated in a multiplicity of biological functions, including energy homeostasis and reproduction. Neuronal systems that are involved in energy homeostasis as well as reproduction traverse the hypothalamus; however, the mechanism by which they control energy homeostasis is not fully understood. The present study analyzes the anatomical relationship of neurons expressing gonadotropin-releasing hormone (GnRH), neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) in a cichlid, tilapia (Oreochromis niloticus). Additionally, we examine in vivo effects of ghrelin on these hypothalamic neurons and plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. Double-immunofluorescence showed neuronal fiber associations between GnRH, NPY and GHRH in the brain and pituitary. Intracerebroventricular injection of ghrelin had no effect on numbers, soma size, or optical density of GnRH and NPY neurons, whereas the number of GHRH neurons was significantly decreased in the animals injected with ghrelin when compared to controls, which may indicate administered ghrelin promoted GHRH release. Plasma GH and pituitary GH mRNA levels were significantly increased in the animals injected with ghrelin. These results suggest that central administration of ghrelin primarily act on hypothalamic GHRH neurons to stimulate GH release from the pituitary in the tilapia.

The present study was conducted to assess an optimal dose of tricaine methansulfonate (MS-222) for rapid anesthesia and recovery in juvenile Yellow perch Perca flavescens. Yellow perch (≈7.0 g/fish) exposed to increasing concentrations of MS-222 (100, 150, 200, 250 and 300 mg/L) showed significant (P<0.05) decreased anesthetic induction and increased recovery time with increasing MS-222 dose. Among the doses tested, the 250 mg/L MS-222 dose showed the shortest anesthetic induction and recovery times. Blood glucose and hematocrit levels were significantly (P<0.05) lower in juvenile yellow perch exposed to the 250 mg/L MS-222 dose than the other MS-222 doses tested. Juvenile yellow perch (about 9.7 g/fish) exposed to 250/mg MS-222 combined with bicarbonate buffer at three ratios (w/w: 1:0.5, 1:1 and 1:2) showed significantly (P<0.05) reduced anesthesia induction and recovery times. The shortest times of anesthesia induction and recover were observed in treatments using the 1:1 MS-222 to buffer ratio. Analysis on serum biochemical parameters (e.g., osmolality, glucose, albumin, calcium, alanine transaminase, total protein and calcium) showed that use of sodium bicarbonate with MS-222 did not have any significant effect on the above measurements compared with those determined in fish exposed to MS-222 only. Anesthesia induction time and recovery time of yellow perch were significantly affected by body sizes. These results suggest that a brief exposure to a 250 mg/L MS-222: sodium bicarbonate dose may be optimal for minimizing handling disturbance in juvenile yellow perch.

This study evaluated the potential of feeding formulated feed to juvenile Lake Sturgeon Acipenser fulvescens and determined the optimal feeding rate of a soft-moist feed on the growth performance and whole-body composition of the fish. Six feeding rates (% body weight [BW]/d) of the soft-moist feed were assigned to each of the 1-week feeding trials: 2%, 4%, 6%, 8%, 10%, and 12%BW/d for trial I; and 0.5%, 2%, 3.5%, 5%, 6.5%, and 8%BW/d for trial II. As reference diets, frozen bloodworms and a dry formulated feed were administered to the fish at feeding rates of 8% and 5%BW/d for trials I and II, respectively. Each treatment was assigned to three tanks, with either 18 fish (trial I; initial BW [mean +/- SD]=3.5 +/- 0.05g) or 15 fish (trial II; initial BW=7.8 +/- 0.1g) per tank. No mortality was observed during the two trials. Weight gain and feed conversation ratio were significantly better for fish that received the soft-moist feed than for those fed the bloodworms. The dry diet and soft-moist diet resulted in similar growth rates. Growth performance and whole-body lipid and moisture content were significantly influenced by feeding rates in both trials. The tyrosine, isoleucine, lysine, phenylalanine, and valine levels in whole fish were significantly altered by the feeding rates in trial I, whereas only the glycine level was altered in trial II. Optimum feeding rates based on the specific growth rate were estimated to be 7.6% and 6.2%BW/d (or 1.47and 1.19MJkgBW(-1)d(-1)) for Lake Sturgeon weighing 3.5 and 7.8g, respectively. These results indicate that Lake Sturgeon can be given formulated feed after previous acclimation to the formulated feed during the early life stage.

ABSTRACT Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression. IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

The purpose of this study was to evaluate the effects of environmentally relevant dietary MeHg exposures on adult female yellow perch (Perca flavescens) and female zebrafish (Danio rerio) ovarian development and reproduction. Yellow perch were used in the study for their socioeconomic and ecological importance within the Great Lakes basin, and the use of zebrafish allowed for a detailed analysis of the molecular effects of MeHg following a whole life-cycle exposure. Chronic whole life dietary exposure of F1 zebrafish to MeHg mimics realistic wildlife exposure scenarios, and the twenty-week adult yellow perch exposure (where whole life-cycle exposures are difficult) captures early seasonal ovarian development. For both species, target dietary accumulation values were achieved prior to analyses. In zebrafish, several genes involved in reproductive processes were shown to be dysregulated by RNA-sequencing and quantitative real-time polymerase chain reaction (QPCR), but no significant phenotypic changes were observed regarding ovarian staging, fecundity, or embryo mortality. Yellow perch were exposed to dietary MeHg for 12, 16, or 20 weeks. In this species, a set of eight genes were assessed by QPCR in the pituitary, liver, and ovary, and no exposure-related changes were observed. The lack of genomic resources in yellow perch hinders the characterization of subtle molecular impacts. The ovarian somatic index, circulating estradiol and testosterone, and ovarian staging were not significantly altered by MeHg exposure in yellow perch. These results suggest that environmentally relevant MeHg exposures do not drastically reduce the reproductively important endpoints in these fish, but to capture realistic exposure scenarios, whole life-cycle yellow perch exposures are needed.

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus, which causes one of the world's most serious fish diseases, infecting >80 freshwater and marine species across the Northern Hemisphere. A new, novel, and especially virulent substrain-VHSv-IVb-first appeared in the Laurentian Great Lakes about a decade ago, resulting in massive fish kills. It rapidly spread and has genetically diversified. This study analyzes temporal and spatial mutational patterns of VHSv-IVb across the Great Lakes for the novel non-virion (Nv) gene that is unique to this group of novirhabdoviruses, in relation to its glycoprotein (G), phosphoprotein (P), and matrix (M) genes. Results show that the Nv-gene has been evolving the fastest (k = 2.0 x 10-3 substitutions/site/year), with the G-gene at ~1/7 that rate (k = 2.8 x 10-4). Most (all but one) of the 12 unique Nv- haplotypes identified encode different amino acids, totaling 26 changes. Among the 12 corresponding G-gene haplotypes, seven vary in amino acids with eight total changes. The P- and M- genes are more evolutionarily conserved, evolving at just ~1/15 (k = 1.2 x 10-4) of the Nv-gene's rate. The 12 isolates contained four P-gene haplotypes with two amino acid changes, and six M-gene haplotypes with three amino acid differences. Patterns of evolutionary changes coincided among the genes for some of the isolates, but appeared independent in others. New viral variants were discovered following the large 2006 outbreak; such differentiation may have been in response to fish populations developing resistance, meriting further investigation. Two 2012 variants were isolated by us from central Lake Erie fish that lacked classic VHSv symptoms, having genetically distinctive Nv-, G-, and M-gene sequences (with one of them also differing in its P-gene); they differ from each other by a G-gene amino acid change and also differ from all other isolates by a shared Nv-gene amino acid change. Such rapid evolutionary differentiation may allow new viral variants to evade fish host recognition and immune responses, facilitating long-time persistence along with expansion to new geographic areas.