Tire-Derived Transformation Product 6PPD-Quinone Induces Mortality and Transcriptionally Disrupts Vascular Permeability Pathways in Developing Coho Salmon.

Urban stormwater runoff frequently contains the car tire transformation product 6PPD-quinone, which is highly toxic to juvenile and adult coho salmon (Onchorychus kisutch). However, it is currently unclear if embryonic stages are impacted. We addressed this by exposing developing coho salmon embryos starting at the eyed stage to three concentrations of 6PPD-quinone twice weekly until hatch. Impacts on survival and growth were assessed. Further, whole-transcriptome sequencing was performed on recently hatched alevin to address the potential mechanism of 6PPD-quinone-induced toxicity. Acute mortality was not elicited in developing coho salmon embryos at environmentally measured concentrations lethal to juveniles and adults, however, growth was inhibited. Immediately after hatching, coho salmon were sensitive to 6PPD-quinone mortality, implicating a large window of juvenile vulnerability prior to smoltification. Molecularly, 6PPD-quinone induced dose-dependent effects that implicated broad dysregulation of genomic pathways governing cell-cell contacts and endothelial permeability. These pathways are consistent with previous observations of macromolecule accumulation in the brains of coho salmon exposed to 6PPD-quinone, implicating blood-brain barrier disruption as a potential pathway for toxicity. Overall, our data suggests that developing coho salmon exposed to 6PPD-quinone are at risk for adverse health events upon hatching while indicating potential mechanism(s) of action for this highly toxic chemical.

Age matters: Comparing life-stage responses to diluted bitumen exposure in coho salmon (Oncorhynchus kisutch).

Millions of liters of diluted bitumen (dilbit), a crude oil product from Canada's oil sands region, is transported through critical Pacific salmon habitat each day. While the toxicity of the water-soluble fraction of dilbit (WSFd) to early life-stages of salmon is known, quantitative data on life-stage differences in sensitivity to WSFd is missing. To fill this knowledge gap, we exposed two juvenile life-stages of coho salmon (O. kisutch) in parallel to very low (parts per billion), environmentally-relevant concentrations of WSFd for acute (48 h) and sub-chronic (4 wk) durations. The relative sensitivities of the two life-stages (fry and parr) were assessed by comparing the timing and magnitude of biological responses using common organismal and molecular endpoints of crude oil exposure. A significant reduction in body condition occurred in both fry and parr after 4 wk exposure to WSFd. Both life-stages also experienced a concentration-dependent decrease in time-to-loss-of-equilibrium during a hypoxia challenge test at both 48 h and 4 wk of exposure. Although organismal responses were similar, molecular responses were distinct between life-stages. In general, unexposed fry had higher baseline values of hepatic phase I biotransformation indicators than unexposed parr, but induction of EROD activity and cyp1a mRNA expression in response to WSFd exposure was greater in parr than in fry. Neither gst nor hsp70 mRNA expression, markers of phase II biotransformation and cell stress, respectively, were reliably altered by WSFd exposure in either life-stage. Taken together, results of this study do not support differential sensitivities of coho fry and parr to WSFd. All the same, the potential for ontogenic differences in the expression and induction of phase I biotransformation need to be considered because age does matter for these endpoints if they are used as bioindicators of exposure in post-spill impact assessments.

Temporal Gene Expression Signature of Plasma Extracellular Vesicles-MicroRNAs from Post-Smolt Coho Salmon Challenged with Piscirickettsia salmonis.

Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.

Sublethal neurotoxicity of organophosphate insecticides to juvenile coho salmon.

For decades, organophosphate (OP) insecticides have been used as chemical control agents in watersheds that support at-risk populations of Pacific salmon throughout western North America. Spray drift, runoff, and other processes transport OPs to critical surface water habitats for migratory salmonids. While most OPs share a common mechanism of action (i.e., inhibition of neuronal acetylcholinesterase, or AChE), they typically vary in toxic potency. Moreover, dose-response relationships for exposure and sublethal neurotoxicity (e.g., brain AChE inhibition) in salmonids have not been defined for many OPs. Here we exposed juvenile coho salmon (Oncorhynchus kisutch) to five common anticholinesterase insecticides (dimethoate, ethoprop, naled, phorate and phosmet) that are widely used on agricultural, commercial, residential, and public lands. Each of the five pesticides produced a concentration-dependent inhibition of AChE enzyme activity. The effective concentration for 50 % AChE inhibition (96-hr EC50) indicated the highest toxicity for phorate (EC50 = 0.57 µg/L) followed by phosmet (3.3 µg/L), naled (7.8 µg/L), ethoprop (90.6 µg/L) and dimethoate (273 µg/L). These findings can inform 1) relative hazard analyses for OP use near sensitive aquatic habitats, 2) predictions of sublethal OP mixture toxicity, and 3) ecological risk assessments for threatened or endangered species of Pacific salmon.

Identification and characterization of miRNAs and lncRNAs of coho salmon (Oncorhynchus kisutch) in normal immune organs.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are two relevant non-coding RNAs (ncRNAs) class. Oncorhynchus kisutch (coho salmon) is an important aquaculture pacific salmon species without report of miRNAs and a very limited register of lncRNAs. To gain knowledge about the interaction and discovery of miRNAs and lncRNAs in coho salmon we used high-throughput sequencing technology to sequence small and transcriptome libraries from three immune organs. A total of 163 mature miRNAs and 4,975 lncRNAs were discovered. The profiles of expression of both ncRNAs indicated that liver and head-kidney share relatively similar expression patterns. We identified 814 and 181 putative target sequences for 1048 lncRNAs and 47 miRNAs, respectively. The results obtained provide new information and enlarge our understanding of the diversities of ncRNAs in coho salmon.

Effect of growth rate on transcriptomic responses to immune stimulation in wild-type, domesticated, and GH-transgenic coho salmon.

BACKGROUND: Transcriptomic responses to immune stimulation were investigated in coho salmon (Oncorhynchus kisutch) with distinct growth phenotypes. Wild-type fish were contrasted to strains with accelerated growth arising either from selective breeding (i.e. domestication) or genetic modification. Such distinct routes to accelerated growth may have unique implications for relationships and/or trade-offs between growth and immune function. RESULTS: RNA-Seq was performed on liver and head kidney in four 'growth response groups' injected with polyinosinic-polycytidylic acid (Poly I:C; viral mimic), peptidoglycan (PGN; bacterial mimic) or PBS (control). These groups were: 1) 'W': wild-type, 2) 'TF': growth hormone (GH) transgenic salmon with ~ 3-fold higher growth-rate than W, 3) 'TR': GH transgenic fish ration restricted to possess a growth-rate equal to W, and 4) 'D': domesticated non-transgenic fish showing growth-rate intermediate to W and TF. D and TF showed a higher similarity in transcriptomic response compared to W and TR. Several immune genes showed constitutive expression differences among growth response groups, including perforin 1 and C-C motif chemokine 19-like. Among the affected immune pathways, most were up-regulated by Poly I:C and PGN. In response to PGN, the c-type lectin receptor signalling pathway responded uniquely in TF and TR. In response to stimulation with both immune mimics, TR responded more strongly than other groups. Further, group-specific pathway responses to PGN stimulation included NOD-like receptor signalling in W and platelet activation in TR. TF consistently showed the most attenuated immune response relative to W, and more DEGs were apparent in TR than TF and D relative to W, suggesting that a non-satiating ration coupled with elevated circulating GH levels may cause TR to possess enhanced immune capabilities. Alternatively, TF and D salmon are prevented from acquiring the same level of immune response as TR due to direction of energy to high overall somatic growth. Further study of the effects of ration restriction in growth-modified fishes is warranted. CONCLUSIONS: These findings improve our understanding of the pleiotropic effects of growth modification on the immunological responses of fish, revealing unique immune pathway responses depending on the mechanism of growth acceleration and nutritional availability.

Growth hormone regulates opsin expression in the retina of a salmonid fish.

Colour vision relies on retinal photoreceptors that express a different predominant visual pigment protein (opsin). In several vertebrates, the primary opsin expressed by a photoreceptor can change throughout ontogeny, although the molecular factors that influence such regulation are poorly understood. One of these factors is thyroid hormone which, together with its receptors, modulates opsin expression in the retinas of multiple vertebrates including rodents and salmonid fishes. In the latter, thyroid hormone induces a switch in opsin expression from SWS1 (ultraviolet light sensitive) to SWS2 (short wavelength or blue light sensitive) in the single cone photoreceptors of the retina. The actions of other hormones on opsin expression have not been investigated. In the present study, we used a transgenic strain of coho salmon (Oncorhynchus kitsutch) with enhanced levels of circulating growth hormone compared to that of wild siblings to assess the effects of this hormone on the SWS1 to SWS2 opsin switch. Transgenic fish showed a developmentally accelerated opsin switch compared to size-matched controls as assessed by immunohistological and in situ hybridisation labelling of photoreceptors and by quantification of transcripts using quantitative polymerase chain reaction. This accelerated switch led to a different spectral sensitivity maximum, under a middle to long wavelength adapting background, from ultraviolet (λmax  ~ 380 nm) in controls to short wavelengths (λmax  ~ 430 nm) in transgenics, demonstrating altered colour vision. The effects of growth hormone over-expression were independent of circulating levels of thyroid hormone (triiodothyronine), the hormone typically associated with opsin switches in vertebrates.

Effect of triploidy on liver gene expression in coho salmon (Oncorhynchus kisutch) under different metabolic states.

BACKGROUND: Triploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild. RESULTS: The vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state. CONCLUSIONS: These findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature.

Proteomic comparison of selective breeding and growth hormone transgenesis in fish: Unique pathways to enhanced growth.

In fish used for food production and scientific research, fast growth can be achieved via selective breeding or induced instantaneously via growth hormone (GH) transgenesis (GHT). The proteomic basis for these distinct routes towards a similar higher phenotype remains uncharacterized, as are associated implications for health parameters. We addressed this knowledge gap using skeletal muscle proteomics in coho salmon (Oncorhynchus kisutch), hypothesising that i) selective breeding and GHT are underpinned by both parallel and unique changes in growth systems, and ii) rapidly-growing fish strains have lowered scope to allocate resources towards immune function. Quantitative profiling of GHT and growth-selected strains was done in comparison to wild-type after injection with PBS (control) or Poly I:C (to mimic infection). We identified remodelling of the muscle proteome in each growth-enhanced strain that was strikingly non-overlapping. GHT was characterized by focal upregulation of systems driving protein synthesis, while the growth-selected fish presented a larger and more diverse set of changes, consistent with complex alterations to many metabolic and cellular pathways. Poly I:C had little detectable effect on the muscle proteome. This study demonstrates that distinct proteome profiles can explain outwardly similar enhanced growth phenotypes, improving our understanding of growth mechanisms in anthropogenic animal strains. SIGNIFICANCE: This work provides the first proteomic insights into mechanisms underpinning different anthropogenic routes to rapid growth in salmon. High-throughput proteomic profiling was used to reveal changes supporting enhanced growth, comparing skeletal muscle of growth hormone transgenic (GHT) and selectively-bred salmon strains with their wild-type counterparts. Contrasting past mRNA-level comparisons of the same fish strains, our data reveals a surprisingly substantial proteomic divergence between the GHT and selectively bred strains. The findings demonstrate that many unique molecular mechanisms underlie growth-enhanced phenotypes in different types of fish strain used for food production and scientific research.

Is the duration of the smolt window related to migration distance in coho salmon Oncorhynchus kisutch?

Physiological changes during the parr-smolt transformation were investigated in short distance (Chilliwack River) and long-distance (Salmon River) migrating coho salmon Oncorhynchus kisutch populations in British Columbia, Canada. Biochemical and molecular indicators were used to monitor smolt development for fish reared at 10 °C throughout the spring. Fish grew well and developed the physical appearance of competent smolts. Both populations exhibited increases in gill Na+ -K+ -ATPase activity (NKA; an important indicator of seawater tolerance) at the same date and the duration of the increase in enzyme activity did not differ between populations. Gill messenger (m)RNA copies for two isoforms of the NKA α subunit, α1a and α1b, showed significant changes and the pattern was similar between populations. Growth hormone receptor and prolactin receptor mRNA from the gill showed modest changes associated with smolting in the spring for both populations, suggesting that these may not be useful indicators of smolt development in hatchery-reared O. kisutch. Consequently, the duration of the smolt window was not based on the region of origin in the present study.

Effect of excessive doses of oxytetracycline on stress-related biomarker expression in coho salmon.

Fish are exposed to a wide variety of environmental stressors, such as chemicals and acute changes in temperature. Oxytetracycline (OTC) has been used as an antibiotic for many kinds of bacterial diseases in cultured fish, but excessive doses of OTC are known to cause side effects in fish and can have negative effects on their environment. In the present study, we examined stress-related biomarker expression in response to excessive doses of dietary OTC in coho salmon (Oncorhynchus kisutch). Fish received OTC (100 mg/kg body weight/day) orally for 2 weeks. The percentage of liver to body weight (hepatosomatic index; HSI) and plasma biochemical parameter, alanine aminotransferase (ALT) activity, of the group fed a diet containing OTC were observed to be significantly higher than those of the control group. The total glutathione (tGSH) levels in the liver of OTC-fed fish were four fold higher than those in control fish and double the control levels in muscle and stomach. Plasma tGSH levels in OTC-fed fish were also higher than those in control fish. Expression levels of heat shock protein 70 in the liver, muscle, and stomach decreased by OTC administration. Accordingly, OTC-induced stress might increase the metabolic turnover of GSH due to consumption by scavenging oxidants generated by stress. These results concerning the changing patterns of stress-related biomarkers indicate that excessive doses of OTC fed to coho salmon induce oxidative stress, which might enhance oxidation in the body and result in damage to tissues, especially in the liver. The present results also suggest that tissue-specific damage caused by OTC might already exist in fish.

In search for globally disordered apo-parvalbumins: Case of parvalbumin ß-1 from coho salmon.

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin ß-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16-19% of all PAs. We show that structural instability of apo-ß-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.

Development of suspect and non-target screening methods for detection of organic contaminants in highway runoff and fish tissue with high-resolution time-of-flight mass spectrometry.

Untreated urban stormwater runoff contributes to poor water quality in receiving waters. The ability to identify toxicants and other bioactive molecules responsible for observed adverse effects in a complex mixture of contaminants is critical to effective protection of ecosystem and human health, yet this is a challenging analytical task. The objective of this study was to develop analytical methods using liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to detect organic contaminants in highway runoff and in runoff-exposed fish (adult coho salmon, Oncorhynchus kisutch). Processing of paired water and tissue samples facilitated contaminant prioritization and aided investigation of chemical bioavailability and uptake processes. Simple, minimal processing effort solid phase extraction (SPE) and elution procedures were optimized for water samples, and selective pressurized liquid extraction (SPLE) procedures were optimized for fish tissues. Extraction methods were compared by detection of non-target features and target compounds (e.g., quantity and peak area), while minimizing matrix interferences. Suspect screening techniques utilized in-house and commercial databases to prioritize high-risk detections for subsequent MS/MS characterization and identification efforts. Presumptive annotations were also screened with an in-house linear regression (log Kowvs. retention time) to exclude isobaric compounds. Examples of confirmed identifications (via reference standard comparison) in highway runoff include ethoprophos, prometon, DEET, caffeine, cotinine, 4(or 5)-methyl-1H-methylbenzotriazole, and acetanilide. Acetanilide was also detected in runoff-exposed fish gill and liver samples. Further characterization of highway runoff and fish tissues (14 and 19 compounds, respectively with tentative identification by MS/MS data) suggests that many novel or poorly characterized organic contaminants exist in urban stormwater runoff and exposed biota.

Uptake and selective partitioning of dietary lipids to ovarian and muscle tissue of maturing female coho salmon, Oncorhynchus kisutch, during secondary oocyte growth.

Female coho salmon, Oncorhynchus kisutch, were fed one of two experimental feeds containing lipids with markedly different stable 13C isotope signatures during the late cortical alveolus, lipid droplet, and vitellogenesis stages of secondary oocyte growth. Ovarian and muscle lipids fatty acid concentrations were significantly affected by treatment during all three stages of development. Stable 13C isotope analyses confirmed that dietary lipids were incorporated into both ovarian and muscle lipids during all three stages and revealed that ovarian lipids were more affected than muscle lipids during vitellogenesis. Arachidonic acid (ARA) was incorporated into ovarian lipids at the highest rate of all fatty acids examined with the greatest uptake observed during the cortical alveolus and lipid droplet stages of development. Docosahexaenoic acid (DHA) was incorporated into ovarian lipids at the next highest rate with the greatest uptake observed during the lipid droplet stage of development. The presence of an ovary specific, fatty acid transfer mechanism is proposed. Results from this study demonstrate the ability to greatly alter the fatty acid composition of ovarian lipids through a dietary change during secondary oocyte growth and may be of great interest to producers of farmed salmon and salmon broodstock programs.

From the Cover: Cadmium Exposure Differentially Alters Odorant-Driven Behaviors and Expression of Olfactory Receptors in Juvenile Coho Salmon (Oncorhynchus kisutch).

Salmon exposed to waterborne metals can experience olfactory impairment leading to disrupted chemosensation. In the current study, we investigated the effects of cadmium (Cd) on salmon olfactory function by modeling an exposure scenario where juvenile salmon transiently migrate through a polluted waterway. Coho were exposed to environmentally relevant concentrations of waterborne Cd (2 and 30 µg/L) for 48 h and (0.3 and 2 µg/L) for 16 days, followed by a 16-day depuration associated with outmigration. Cadmium exposures inhibited behavioral responses towards L-cysteine and conspecific odorants, with effects persisting following the depuration. Behavioral alterations following the 30 µg/L exposure were associated with increased olfactory epithelial gene expression of metallothionein (mt1a) and heme oxygenase (hmox1); reduced expression of olfactory signal transduction (OST) molecules; and reduced expression of mRNAs encoding major coho odorant receptors (ORs). Salmon OR array analysis indicated that Cd preferentially impacted expression of OST and OR markers for ciliated olfactory sensory neurons (OSNs) relative to microvillus OSNs, suggesting a differential sensitivity of these two major OSN populations. Behavioral alterations on exposure to 0.3 and 2 µg/L Cd were associated with increased mt1a, but not with major histological or OR molecular changes, likely indicating disrupted OST as a major mechanism underlying the behavioral dysfunction at the low-level Cd exposures. Laser-ablation mass spectrometry analysis revealed that the OSN injury and behavioral dysfunction was associated with significant Cd bioaccumulation within the olfactory sensory epithelium. In summary, low-level Cd exposures associated with polluted waterways can induce differential and persistent olfactory dysfunction in juvenile coho salmon.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium.

Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia.

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity.

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors.

RNAseq analysis of fast skeletal muscle in restriction-fed transgenic coho salmon (Oncorhynchus kisutch): an experimental model uncoupling the growth hormone and nutritional signals regulating growth.

BACKGROUND: Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. RESULTS: Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. CONCLUSIONS: TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling.

Food Shortage Causes Differential Effects on Body Composition and Tissue-Specific Gene Expression in Salmon Modified for Increased Growth Hormone Production.

Growth hormone (GH) transgenic salmon possesses markedly increased metabolic rate, appetite, and feed conversion efficiency, as well as an increased ability to compete for food resources. Thus, the ability of GH-transgenic fish to withstand periods of food deprivation as occurs in nature is potentially different than that of nontransgenic fish. However, the physiological and genetic effects of transgenic GH production over long periods of food deprivation remain largely unknown. Here, GH-transgenic coho salmon (Oncorhynchus kisutch) and nontransgenic, wild-type coho salmon were subjected to a 3-month food deprivation trial, during which time performance characteristics related to growth were measured along with proximate compositions. To examine potential genetic effects of GH-transgenesis on long-term food deprivation, a group of genes related to muscle development and liver metabolism was selected for quantitative PCR analysis. Results showed that GH-transgenic fish lose weight at an increased rate compared to wild-type even though proximate compositions remained relatively similar between the groups. A total of nine genes related to muscle physiology (cathepsin, cee, insulin-like growth factor, myostatin, murf-1, myosin, myogenin, proteasome delta, tumor necrosis factor) and five genes related to liver metabolism (carnitine palmitoyltransferase, fatty acid synthase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glucokinase) were shown to be differentially regulated between GH-transgenic and wild-type coho salmon over time. These genetic and physiological responses assist in identifying differences between GH-transgenic and wild-type salmon in relation to fitness effects arising from elevated growth hormone during periods of long-term food shortage.