Ghrelin Modulates Differential Expression of Genes Relevant to Immune Activities and Antimicrobial Peptides in Primary Head Kidney Cells of Rainbow Trout (Oncorhynchus mykiss).

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.

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1.

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.

Molecular Analysis and Sex-specific Response of the Hepcidin Gene in Yellow Perch (Perca Flavescens) Following Lipopolysaccharide Challenge.

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.

Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss).

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 (~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.

Comparative effects of Novirhabdovirus genes on modulating constitutive transcription and innate antiviral responses, in different teleost host cell types.

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 ß-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.

Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway.

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.

Ghrelin stimulates growth hormone release from the pituitary via hypothalamic growth hormone-releasing hormone neurons in the cichlid, Oreochromis niloticus.

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.

Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver.

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-1ß: 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-1ß 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-1ß, 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.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

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.

Gene Diversification of an Emerging Pathogen: A Decade of Mutation in a Novel Fish Viral Hemorrhagic Septicemia (VHS) Substrain since Its First Appearance in the Laurentian Great Lakes.

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.

Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): enhanced quality control with internal standards.

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

Characterization and evaluation of sex-specific expression of suppressors of cytokine signaling (SOCS)-1 and -3 in juvenile yellow perch (Perca flavescens) treated with lipopolysaccharide.

The suppressor of cytokine signaling (SOCS) proteins are a family of intracellular proteins that are centrally involved with vertebrate growth, development and immunity via their effects as negative feed-back regulators of cytokine (and hormone) signaling. The genes for SOCS-1 & -3 were cloned, sequences analyzed and expression patterns examined in the commercially-important teleost, yellow perch (Perca flavescens). The deduced (mature) proteins for yellow perch (yp)SOCS-1 and (yp)SOCS-3 consist of 211 and 205 amino acids, respectively. Functional domains such as the Src homology-2 (SH2) and SOCS-box were present in ypSOCS-1 and ypSOCS-3 and these domains were well conserved between teleost species. Sequence analysis showed that ypSOCS-1 & -3 share highest homology (among similar teleost sequences), to the stickleback (Gasterosteus aculatus) SOCS-1 & -3 protein homologs. To investigate sex-specific expression of the ypSOCS-1 and ypSOCS-3 mRNAs, juvenile male and female yellow perch were immunologically challenged with a single injection (10 µg/g bw) of lipopolysaccharide (LPS) and tissues (gill, head kidney, kidney, liver and spleen) were sampled over a 48-h time-course. Quantitative real-time PCR analysis showed that ypSOCS-1 & -3 were expressed in all tissues examined and at all sampling time-points. LPS injection significantly induced ypSOCS-1 & -3 mRNA levels in gill, head kidney, liver, kidney and spleen, with maximal induction occurring at 6 h post-injection in each tissue. By 48-h post-injection, expression levels for ypSOCS-1 & -3 mRNAs approached, or reached, control levels in all tissues examined. While there were statistical interactions among variables (treatment, time and sex) for ypSOCS-1, we only found a main effect of sex on SOCS-3 mRNA expression in head kidney with higher copy numbers occurring in males than in females treated with LPS. Sexually-dimorphic expression of SOCS-1 or -3 mRNA has not been examined, or described, in a teleost. Our findings suggest the involvement of the SOCS genes in the yellow perch immune response and that differences among the sexes are evident and should be explored further.

Developmental expression and estrogen responses of endocrine genes in juvenile yellow perch (Perca flavescens).

The present study examines the expression of growth-regulating genes (gh, prl, smtl and igf1b), the estrogen receptors (esr1 and esr2a) and aromatase (cyp19a1a) in developing yellow perch. To gain an initial understanding into the endocrine control of growth preceding and involved with sexual size dimorphism (SSD), where females have been reported to grow faster and larger than males, young of the year fish were sampled for length, weight and tissues at several time points (102-421 days post-hatch (dph)). Positive growth was seen in both sexes over the sampling interval, but SSD was not manifested. Using real-time quantitative PCR, we found that pituitary growth hormone (gh) and liver insulin-like growth factor-1b (igf1b) mRNA levels were significantly affected by dph and levels were found to be correlated with growth in both sexes. Liver cyp19a1a, esr1 and esr2a mRNA levels were significantly influenced by dph, whereas there was a significant dph*sex interaction on liver esr2a mRNA levels with males having higher levels than females at 379 and 421 dph. Ovarian cyp19a1a decreased with dph, but there were no changes in esr1 or esr2a mRNA levels. Dietary treatment of juvenile (∼300 dph) females with 20 mg/kg diet 17ß-estradiol resulted in significantly higher liver esr1 mRNA levels and a sustained hepatosomatic index (I(H)). Across all data sets liver esr2a mRNA levels showed the most significant positive correlation with liver igf1b mRNA levels. These findings show that growth is accompanied by increases in pituitary gh, liver igf1b and liver esr1 and esr2a mRNAs in juvenile yellow perch.

Stimulation of growth and changes in the hepatic transcriptome by 17beta-estradiol in the yellow perch (Perca flavescens).

The effects of dietary 17beta-estradiol (E(2)) on growth and liver transcriptomics were investigated in the yellow perch (Perca flavescens). After a 3-mo treatment, E(2) significantly stimulated an increase in length and weight of juvenile male and female perch relative to control animals. The increase was significantly greater in females compared with males. Separate, unnormalized cDNA libraries were constructed from equal quantities of RNA from 6 male and 6 female livers of E(2)-treated and control perch, and 3,546 and 3,719 expressed sequence tags (ESTs) were obtained, respectively. To characterize E(2)-regulated transcripts, EST frequencies between libraries were calculated within contiguous sequences that were assembled from the combined ESTs of both libraries. Frequencies were also determined in EST transcript groupings produced by aligning all of the ESTs from both libraries at the nucleotide level. From these analyses, there were 28 annotated transcripts that were regulated by 75% between libraries and for which there were at least 5 ESTs of the same transcript between libraries. Regulation of a subset (14) of these transcripts was confirmed by quantitative reverse transcription-polymerase chain reaction (QPCR). Transcripts that were upregulated by E(2) included reproduction-related proteins, binding proteins, and proteases and protease inhibitors. While not part of the transcript frequency analysis, QPCR showed significant upregulation of estrogen receptor esr1 and of insulin-like growth factor I (IGF-I) in E(2) livers. E(2)-downregulated transcripts represented a variety of functional categories including components of the respiratory chain, lipid transport and metabolism, glycolysis, amino acid and nitrogen metabolism, binding proteins, a hydrolytic enzyme, and a transcriptional regulator. In perch it appears that exogenous estrogen drastically shifts liver metabolism toward the production of lipoproteins and carbohydrate binding proteins, and that the growth-promoting action may involve an increase in hepatic IGF-I production.

Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss).

Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects of Posilac (Monsanto, St. Louis, MO), a recombinant bovine GH, on tissue transcript levels in rainbow trout selected from two high-growth rate and two low-growth rate families. Transcript abundance was measured in liver and muscle with the Genome Research in Atlantic Salmon Project (GRASP) 16K cDNA microarray. A selection of the genes identified as altered by the microarray and transcripts for insulin-like growth factors, growth hormone receptors (GHRs), and myostatins were measured by real-time PCR in the liver, muscle, brain, kidney, intestine, stomach, gill, and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar directional changes of expression in the other nonhepatic tissues. A total of 114 and 66 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver, respectively. The largest proportion of these transcripts represented novel transcripts, followed by immune and metabolism-related genes. We have identified a number of genes related to lipid metabolism, supporting a modulation in lipid metabolism following GH treatment. Most notable among the growth-axis genes measured by real-time PCR were increases in GHR1 and -2 transcripts in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors, and modulates growth-regulating genes.

Molecular characterization and sex-specific tissue expression of estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens).

Yellow perch (Perca flavescens) exhibits an estrogen-stimulated sexual size dimorphism (SSD) wherein females grow faster and larger than males. To aid in the examination of this phenomenon, the cDNA sequences encoding estrogen receptor-alpha (esr1), estrogen receptor-betaa (esr2a) and ovarian aromatase (cyp19a1a) for the teleost yellow perch were obtained. Several tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNAs of yellow perch esr1, esr2a and cyp19a1a consist of 3052 bp, 2462 bp and 1859 bp with open reading frames encoding putative proteins of 576 amino acids, 555 amino acids and 518 amino acids, respectively. Esr1 and esr2a expression was highest in female ovary and liver tissues with low to moderate expression in other tissues. Esr2a showed a more global tissue expression pattern than esr1, particularly in males but also in females. Cyp19a1a expression was highest in both male and female spleen tissue and oocytes with moderate expression in male pituitary and gill tissue. Cyp19a1a expression was moderately high in female liver tissue with undetectable expression in male liver tissue, suggesting its involvement in sexually dimorphic growth. These sequences are valuable molecular tools that can be used in future studies investigating estrogen mechanisms and actions, such as SSD, in yellow perch.

Molecular characterization and sex-specific tissue expression of prolactin, somatolactin and insulin-like growth factor-I in yellow perch (Perca flavescens).

The cDNA sequences encoding prolactin (PRL), somatolactin (SL) and insulin-like growth factor-I (IGF-I) genes of the yellow perch were obtained. Brain, pituitary, gill, heart, liver, stomach, kidney, spleen, muscle and gonad tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNA of yellow perch PRL consists of 2306 bp and PRL expression was highest in the yellow perch pituitary with low to moderate expression in other tissues including brain, gill and post-vitellogenic oocytes. The full length cDNA of yellow perch SL consists of 1589 bp and SL expression was highest in the yellow perch pituitary with low to moderate expression in other tissues including brain, gill, liver, stomach, spleen and kidney. The full length cDNA of yellow perch IGF-Ib consists of 814 bp and tissue expression analysis of yellow perch IGF-I revealed a second yellow perch transcript (IGF-Ia) that is 81 nucleotides smaller. Both IGF-Ib and IGF-Ia had the greatest expression in liver tissue with moderate expression in brain, spleen and kidney tissues of both sexes. These sequences are valuable molecular tools which can be used in future studies investigating the basis for sexually dimorphic growth in yellow perch.

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss).

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus): effect of seawater exposure and cortisol treatment.

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.