Modeling the toxicity of dissolved crude oil exposures to characterize the sensitivity of cod (Gadus morhua) larvae and role of individual and unresolved hydrocarbons.

Toxicity of weathered oil was investigated using Atlantic cod (Gadus morhua) larvae. A novel exposure system was applied to differentiate effects associated with dissolved and droplet oil with and without dispersant. After a 4-day exposure and subsequent 4-day recovery period, survival and growth were determined. Analytical data characterizing test oil composition included polyaromatic hydrocarbons (PAH) based on GC/MS and unresolved hydrocarbon classes obtained by two-dimensional chromatography coupled with flame ionization detection was used as input to an oil solubility model to calculate toxic units (TUs) of dissolved PAHs and whole oil, respectively. Critical target lipid body burdens derived from modeling characterizing the sensitivity of effect endpoints investigated were consistent across treatments and within the range previously reported for pelagic species. Individually measured PAHs captured only 3-11% of the TUs associated with the whole oil highlighting the limitations of traditional total PAH exposure metrics for expressing oil toxicity data.

Combined effects of fishing and oil spills on marine fish: Role of stock demographic structure for offspring overlap with oil.

It has been proposed that the multiple pressures of fishing and petroleum activities impact fish stocks in synergy, as fishing-induced demographic changes in a stock may lead to increased sensitivity to detrimental effects of acute oil spills. High fishing pressure may erode the demographic structure of fish stocks, lead to less diverse spawning strategies, and more concentrated distributions of offspring in space and time. Hence an oil spill may potentially hit a larger fraction of a year-class of offspring. Such a link between demographic structure and egg distribution was recently demonstrated for the Northeast Arctic stock of Atlantic cod for years 1959-1993. We here estimate that this variation translates into a two-fold variation in the maximal proportion of cod eggs potentially exposed to a large oil spill. With this information it is possible to quantitatively account for demographic structure in prospective studies of population effects of possible oil spills.

Diet affects the redox system in developing Atlantic cod (Gadus morhua) larvae.

The growth and development of marine fish larvae fed copepods is superior to those fed rotifers, but the underlying molecular reasons for this are unclear. In the following study we compared the effects of such diets on redox regulation pathways during development of Atlantic cod (Gadus morhua) larvae. Cod larvae were fed a control diet of copepods or the typical rotifer/Artemia diet commonly used in commercial marine fish hatcheries, from first feeding until after metamorphosis. The oxidised and reduced glutathione levels, the redox potential, and the mRNA expression of 100 genes in redox system pathways were then compared between treatments during larval development. We found that rotifer/Artemia-fed cod larvae had lower levels of oxidised glutathione, a more reduced redox potential, and altered expression of approximately half of the redox system genes when compared to copepod-fed larvae. This rotifer/Artemia diet-induced differential regulation of the redox system was greatest during periods of suboptimal growth. Upregulation of the oxidative stress response transcription factor, nrf2, and NRF2 target genes in rotifer/Artemia fed larvae suggest this diet induced an NRF2-mediated oxidative stress response. Overall, the data demonstrate that nutritional intake plays a role in regulating the redox system in developing fish larvae. This may be a factor in dietary-induced differences observed in larval growth.

Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua).

For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod.

Substitution of fish oil with camelina oil and inclusion of camelina meal in diets fed to Atlantic cod (Gadus morhua) and their effects on growth, tissue lipid classes, and fatty acids.

Developing a commercially relevant Atlantic cod aquaculture industry will require improvements in feed sustainability. Camelina oil and meal are potential replacements of fish oil and fish meal in aquaculture feeds. Camelina oil is high in 18:3ω3 (30%), with an ω3/ω6 ratio > 1. Camelina meal has a considerable crude protein level (38%), which includes significant amounts of methionine and phenylalanine. Four diets were tested; each diet was fed to triplicate tanks (3 tanks per diet) of Atlantic cod (14.4 g/fish; 70 fish per tank) for 13 wk. The diets included a fish oil/fish meal control (FO) and three diets which replaced 100% of fish oil with camelina oil: one diet contained fish meal (100CO), another solvent extracted fish meal (100COSEFM), and another had fish meal partially reduced by 15% inclusion of camelina meal (100CO15CM). Growth was measured (length and weight) and tissue samples were collected for lipid analysis (muscle, liver, brain, gut, spleen, skin, and carcass) at wk 0 (before feeding the experimental diet) and at wk 13. Cod fed camelina oil had a lower (P < 0.001) final weight than cod fed the FO diet (50.8 ± 10.3 g/fish). Cod fed 100CO15CM had a lower (P < 0.001) final weight (35.0 ± 8.0 g) than those fed 100CO (43.6 ± 8.9 g) and 100COSEFM (46.7 ± 10.7 g). Cod tissues in the 100COSEFM treatment were most impacted by dietary fatty acid profile. Multivariate statistics revealed that FO and 100COSEFM tissue fatty acid profiles were 21 to 31% different, depending on tissue type. The full replacement of fish oil with camelina oil, plus solvent extracted fish meal had an overarching effect on the entire fatty acid profile of the whole animal. Fatty acid mass balance calculations indicated that cod fed 100COSEFM elongated 13% of 18:3ω3 to 20:3ω3 and oxidized the remaining 87%, whereas cod fed fish oil showed a much lower (P < 0.001) elongation of 18:3ω3 of 1.6%. These results suggest that excess 18:3ω3 from camelina oil caused some fatty acid elongation, but little desaturation. Energy budget estimates indicated that cod fed 100COSEFM deposited the most energy throughout the trial (60 kJ/fish; P = 0.019), mostly in the liver (90%). Excess camelina lipids were not necessarily utilized for energy, which likely impacted growth. Feeding 100% camelina oil to Atlantic cod impacted growth and lipid and fatty acid composition; however, additional removal of fish oil from fish meal caused the greatest change in cod lipid composition and utilization.

Electrophysiological differences between fshb- and lhb-expressing gonadotropes in primary culture.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²âº responses were similar. Expression of Ca²âº-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²âº influx.

Effect of replacement of fish oil with camelina (Camelina sativa) oil on growth, lipid class and fatty acid composition of farmed juvenile Atlantic cod (Gadus morhua).

Camelina (Camelina sativa) oil was tested as a replacement for fish oil in diets for farmed Atlantic cod (Gadus morhua). Camelina differs from other plant oilseeds previously used in aquaculture with high lipid (40 %), α-linolenic acid (40 %), antioxidants and low proportions of saturated fats. Dietary treatments were fed to cod (19 g fish⁻¹ initial weight) for 9 weeks and included a fish oil control (FO), 40 % (CO40) and 80 % (CO80) replacement of fish oil with camelina oil. There was no effect of replacing fish oil with camelina oil included at levels up to 80 % on the growth performance. Cod fed CO80 stored more lipid in the liver (p < 0.01), including more neutral lipid (p < 0.05) and triacylglycerol (p < 0.05). Cod fed CO80 decreased in total polyunsaturated fatty acids (PUFAs) in muscle compared to CO40 and FO (p < 0.05), increased in monounsaturated fatty acids (p < 0.01), decreased in total ω3 fatty acids (FO > CO40 > CO80; p < 0.01) and increased in total ω6 fatty acids (FO < CO40 < CO80; p < 0.01). In the liver, long-chain (LC) PUFA such as 20:4ω6, 20:5ω3, 22:5ω3 and 22:6ω3 decreased when fish oil was removed from the diet (p < 0.05), and increased in 18-carbon fatty acids (p < 0.01). Camelina oil can reduce the amount of fish oil needed to meet lipid requirements, although replacing 80 % of fish oil reduced LC PUFAs in both tissues. A comparison of BF3 and H2SO4 as catalysts to transmethylate cod liver and muscle lipids revealed small but significant differences in some fatty acid proportions.

Localization and functional properties of two galectin-1 proteins in Atlantic cod (Gadus morhua) mucosal tissues.

Galectin-1 is a ß-galactoside binding lectin with multiple immune functions in higher vertebrates. We report the characterization of two galectin-1 proteins from Atlantic cod, with emphasis on mucosal tissues. Tissue distribution of these two ≈14kDa galectin-1 proteins (Codgal1-1 and Codgal1-2) was ascertained by western blotting of one dimensional (1D) and two dimensional (2DE) gels. The two galectin-1 proteins were differentially localized in the mucosal tissues of cod. Codgal1-1 was predominantly localized in the basal cells of skin and this protein was present in all the early developmental stages examined, indicating a likely involvement in developmental processes. The two lectins were also localized in the adherent macrophage-like cells (MLC) from cod head kidney and results gathered indicate their possible secretion during Francisella noatunensis infection, suggesting that they are active components of immune defence. Lactose affinity chromatography coupled with gel filtration co-purified the two cod galectin-1 proteins, which hemagglutinated horse red blood cells in a lactose inhibitable manner. They also could bind and agglutinate both Gram-positive and Gram-negative bacteria. This study suggests multiple functional roles for galectin-1, especially in development and innate immune response of Atlantic cod.

Photoperiod influences growth and mll (mixed-lineage leukaemia) expression in Atlantic cod.

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species.

Photoperiod effects on the expression of kisspeptin and gonadotropin genes in Atlantic cod, Gadus morhua, during first maturation.

In order to investigate the potential role of the kisspeptin system in the entrainment of reproduction in Atlantic cod, qPCR assays were developed for kiss2 and its receptor kissr4. mRNA expression was monitored in the brain over a full reproductive cycle in 2 populations of males and females: 1) a maturing population (exposed to simulated natural photoperiod, SNP) and 2) a maturation inhibited population (exposed to constant light, LL). Pituitary expression of gonadotropin subunit mRNA (fshß and lhß) was also measured. Results from this study indicated no clear temporal pattern in expression of kiss2 or kissr4 mRNAs in either population of cod, however acute elevations were apparent in maturing (SNP) individuals, namely an elevation in kiss2 in vitellogenic females and spermiating males and spikes in kissr4 during early vitellogenesis in females and spermatogenesis in males. Gonadotropin mRNA expression displayed strong amplitudinal changes over time with fshß and lhß mRNA expression increasing towards spawning in maturing individuals. No firm conclusions on the role of the kisspeptin system in cod puberty can be drawn at this stage, however mRNA increases in kiss2 and kissr4 may elude to conserved kisspeptin functions in cod and opens up interesting avenues on potential gender specific functions.

Transcriptomic analyses of intestinal gene expression of juvenile Atlantic cod (Gadus morhua) fed diets with Camelina oil as replacement for fish oil.

For aquaculture of marine species to continue to expand, dietary fish oil (FO) must be replaced with more sustainable vegetable oil (VO) alternatives. Most VO are rich in n-6 polyunsaturated fatty acids (PUFA) and few are rich in n-3 PUFA but Camelina oil (CO) is unique in that, besides high 18:3n-3 and n-3/n-6 PUFA ratio, it also contains substantial long-chain monoenes, commonly found in FO. Cod (initial mass ~1.4 g) were fed for 12 weeks diets in which FO was replaced with CO. Growth performance, feed efficiency and biometric indices were not affected but lipid levels in liver and intestine tended to increase and those of flesh, decrease, with increasing dietary CO although only significantly for intestine. Reflecting diet, tissue n-3 long-chain PUFA levels decreased whereas 18:3n-3 and 18:2n-6 increased with inclusion of dietary CO. Dietary replacement of FO by CO did not induce major metabolic changes in intestine, but affected genes with potential to alter cellular proliferation and death as well as change structural properties of intestinal muscle. Although the biological effects of these changes are unclear, given the important role of intestine in nutrient absorption and health, further attention should be given to this organ in future.

Oil droplets do not affect assimilation and survival probability of first feeding larvae of North-East Arctic cod.

Oil exploration and production in the Atlantic moves northwards towards spawning and nursery areas of fish species that sustain some of the world's largest fisheries. Models are therefore needed that can simulate the effects of accidental oil spills on early life stages of these fish. In this study, we combined an individual based model and a microcosm approach to infer effects of the water soluble fraction (WSF) and of an oil dispersion (WSF and droplets) on two key endpoints of North East Arctic cod (Gadus morhua) larvae: food assimilation rate and survival probability. Both exposure types (WSF and dispersion) decreased assimilation rate (control: 0.4 d(-1)) and survival probability (control: 0.96) in a concentration-dependent fashion, with EC(50)s of about 2 (feeding) and 40 µg/L ∑PAH in the WSF (survival probability). No consistent differences were found between the ECs from the two exposure types indicating no additional oil droplet effects in the oil dispersion. During post exposure, effects on the two endpoints disappeared, which was confirmed by an image analyses we performed of gut content fluorescence. Our results also show that the larvae model fitted the experimental data from the two exposure types equally well, indicating that the presence of oil droplets did not affect model performance. More complex models that explicitly consider possible mechanisms of oil droplet toxicity - in addition to the toxicity of the WSF - on the two examined endpoints during a 17 day time frame do therefore not have a higher accuracy than simpler models that neglect oil droplet toxicity.

Transcriptional evidence for low contribution of oil droplets to acute toxicity from dispersed oil in first feeding Atlantic cod (Gadus morhua) larvae.

We evaluated the potential contribution of oil droplets to the toxicity of dispersed oil to first feeding fish larvae. Atlantic cod larvae were exposed to five concentrations of either artificially weathered (200°C residue) dispersed oil (D1-D5) containing oil droplets [medium size 11-13 µm based on volume] and water-soluble fraction [WSF] or the filtered dispersions containing only the corresponding equilibrium WSFs only (W1-W5). The larvae were exposed for 4 days and harvested for transcriptional analysis at 13 days post hatching. The most significant differently expressed genes were observed in cod larvae exposed to the highest concentration of the dispersed oil (containing 10.41 ± 0.46 µg ∑PAH/L), with CYP1A showing the strongest response. Functional analysis further showed that the top scored network as analyzed with Ingenuity Pathway Analysis was "Drug Metabolism, Endocrine System Development and Function, Lipid Metabolism". Oil exposure also increased the expression of genes involved in bone resorption and decreased the expression of genes related to bone formation. In conclusion, oil exposure affects drug metabolism, endocrine regulation, cell differentiation and proliferation, apoptosis, fatty acid biosynthesis and tissue development in Atlantic cod larvae. The altered gene transcription was dominated by the WSF and the corresponding oil droplet fraction only had a moderate contribution to the observed changes.

Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish.

Gonadotropin releasing hormone (GnRH) is a key regulator of sexual development and reproduction in vertebrates. Fish have either two or three pre-pro-GnRH genes, encoding structurally distinct peptides. We identified three pre-pro-GnRH genes in Atlantic cod (Gadus morhua, gmGnRH) using RT-PCR, RACE-PCR and BAC DNA library clone sequencing based on synteny searching. Gene identity was confirmed by sequence alignment and subsequent phylogenetic analysis. The expression of these genes was measured by quantitative PCR in the brain and pituitary of female cod throughout their reproductive cycle and in peripheral tissues. All three gmGnRH genes have highly conserved deduced decapeptide sequences, but sequence and phylogenetic data for gmGnRH1 suggest that this is a pseudogene. gmGnRH1 shares low identity with all fish GnRH variants and grouped with the GnRH3 clade. Although gmGnRH1 is a putative pseudogene, it is transcribed in multiple tissues but at low levels in the brain, indicating the loss of conserved hypophysiotrophic function. Phylogenetic analysis reveals that gmGnRH2 and gmGnRH3 variants are located in variant-specific clades. Both gmGnRH2 and gmGnRH3 transcripts are most abundant in the brain, with lower expression in pituitaries and ovaries. Brain gmGnRH3 gene expression increases in spawning fish and is expressed in the pituitary during puberty. Brain gmGnRH2 transcripts are highly expressed relative to gmGnRH3 before and during spawning. Sequence and expression data suggest that gmGnRH1 is a pseudogene and that gmGnRH3 is likely the hypophysiotrophic form of GnRH in Atlantic cod.

Ecotoxicological mechanisms and models in an impact analysis tool for oil spills.

In an international collaborative effort, an impact analysis tool is being developed to predict the effect of accidental oil spills on recruitment and production of Atlantic cod (Gadus morhua) in the Barents Sea. The tool consisted of three coupled ecological models that describe (1) plankton biomass dynamics, (2) cod larvae growth, and (3) fish stock dynamics. The discussions from a series of workshops are presented in which variables and parameters of the first two ecological models were listed that may be affected by oil-related compounds. In addition, ecotoxicological algorithms are suggested that may be used to quantify such effects and what the challenges and opportunities are for algorithm parameterization. Based on model exercises described in the literature, survival and individual growth of cod larvae, survival and reproduction of zooplankton, and phytoplankton population growth are denoted as variables and parameters from the ecological models that might be affected in case of an oil spill. Because toxicity databases mostly (67%) contain data for freshwater species in temperate environments, parameterization of the ecotoxicological algorithms describing effects on these endpoints in the subarctic marine environment is not straightforward. Therefore, it is proposed that metadata analyses be used to estimate the sensitivity of subarctic marine species from available databases. To perform such analyses and reduce associated uncertainty and variability, mechanistic models of varying complexity, possibly aided by new experimental data, are proposed. Lastly, examples are given of how seasonality in ecosystems may influence chemical effects, in particular in the subarctic environment. Food availability and length of day were identified as important characteristics as these determine nutritional status and phototoxicity, respectively.

Transcriptional effects on glutathione S-transferases in first feeding Atlantic cod (Gadus morhua) larvae exposed to crude oil.

Polycyclic aromatic hydrocarbons (PAHs) and other oil compounds are known to induce stress and impact health of marine organisms. Water-soluble fractions of oil contain components known to induce glutathione S-transferases (GSTs), one of the major classes of phase II detoxifying enzymes present in essentially all eukaryotic organisms. In this study, the transcriptional responses of six GSTs (GST pi, GST mu, GST omega, GST theta, GSY zeta and GST kappa) were examined in early larvae of Atlantic cod Gadus morhua exposed to five concentrations of dispersed oil (containing oil droplets and water-soluble fraction) and water-soluble fractions (WSF) of oil. When Atlantic cod larvae were exposed to WSF (containing 1.31+/-0.31microg summation PAH/L for 4 days), expression of GSTM3 and GSTO1 was significantly increased, whereas no differences in GST expression were observed in larvae exposed to a corresponding 50% lower amount of dispersed oil (containing 0.36+/-0.10 microg summation PAH/L for 4 days). The study suggest that although the oil clearly had severe negative effects on the larvae (i.e. concentration-dependent lethality and growth reduction), only minor effects on GST transcription could be observed using RNA obtained from pooled whole-larvae homogenates. This result indicates that the expression of these important detoxification enzymes is only moderately inducible at such an early developmental stage either reflecting low tolerance of cod larvae to dispersed oil or alternatively that using whole-larvae homogenates may have masked tissue-specific mRNA induction.

Ontogenetic effects of diet during early development on growth performance, myosin mRNA expression and metabolic enzyme activity in Atlantic cod juveniles reared at different salinities.

This study investigates the effect of diet during early development on growth and metabolic capacity in the juvenile stage of Atlantic cod. Growth in three groups of Atlantic cod juveniles (10-70 g) was measured at two salinities (15 per thousand or 32 per thousand) in combination with two temperatures (10 degrees C or 14 degrees C). Groups of cod from a single egg batch differed by having been fed with rotifers (R) or natural zooplankton (Z) during the first 36 days post hatch. A third group was fed zooplankton from 1 to 22 dph, after which diet changed to rotifers from 22 to 36 dph (ZRZ). All fish were weaned at 36 dph. Juveniles from the Z and ZRZ groups performed equally well under all experimental conditions, but fish that had received rotifers as a larval diet showed overall significantly lower growth rates. Growth was significantly enhanced by reduced salinity. Metabolic enzyme activity and relative myosin mRNA expression levels were not affected by larval diet. Muscle AAT and MDH were affected by salinity while these enzymes in liver tissue were affected by the interaction between salinity and temperature. Metabolic enzymes were stronger correlated with fish size than growth rates. Our results indicate that larval diet has a pronounced effect on juvenile growth rates under varying environmental conditions as optimal larval diet (zooplankton) increased juvenile growth rates significantly. Metabolic enzyme activity and relative myosin mRNA expression were not affected by larval history, which suggests that the persisting juvenile growth difference is not a result of differing metabolic capacity.

Biomarker candidate discovery in Atlantic cod (Gadus morhua) continuously exposed to North Sea produced water from egg to fry.

In this study Atlantic cod (Gadus morhua) were exposed to different levels of North Sea produced water (PW) and 17beta-oestradiol (E(2)), a natural oestrogen, from egg to fry stage (90 days). By comparing changes in protein expression following E(2) exposure to changes induced by PW treatment, we were able to compare the induced changes by PW to the mode of action of oestrogens. Changes in the proteome in response to exposure in whole cod fry (approximately 80 days post-hatching, dph) were detected by two-dimensional gel electrophoresis and image analysis and identified by MALDI-ToF-ToF mass spectrometry, using a newly developed cod EST database and the NCBI database. Many of the protein changes occurred at low levels (0.01% and 0.1% PW) of exposure, indicating putative biological responses at lower levels than previously detected. Using discriminant analysis, we identified a set of protein changes that may be useful as biomarker candidates of produced water (PW) and oestradiol exposure in Atlantic cod fry. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring exposure and effects of discharges from the petroleum industry offshore, aiding future environmental risk analysis and risk management.

Quantification of gonadotropin subunits GPalpha, FSHbeta, and LHbeta mRNA expression from Atlantic cod (Gadus morhua) throughout a reproductive cycle.

To elucidate the role of the gonadotropins in Atlantic cod (Gadus morhua), complete coding sequences with partially or fully un-translated regions for the three subunits GPalpha, FSHbeta, and LHbeta were determined. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-targeting quantitative PCR assays. Relative expression was analyzed during a complete seasonal sexual maturation cycle in Atlantic cod females. Increasing levels of lhbeta mRNA were observed during gonadal growth, peaking at spawning in February-March which corresponds to maximum gonadosomatic index. In contrast, both gpalpha and fshbeta gradually increased to a peak in December, two months before spawning started, and decreased in January just prior to spawning. Both mRNAs increased again and remained high during the spawning season, with a decline at the end of the spawning period, a further decrease in spent females, followed by a new gradual increase concurrent with the start of the next reproductive cycle. In addition to its role in vitellogenesis prior to spawning, FSH seems to have additional functions during the spawning period, possibly related to vitellogenesis that runs in parallel with final oocyte maturation and ovulation of the multiple batch spawner Atlantic cod.