Article pubs.acs.org/est
Distinctive Metabolite Profiles in In-Migrating Sockeye Salmon Suggest Sex-Linked Endocrine Perturbation Jonathan P. Benskin,*,†,‡ Michael G. Ikonomou,‡ Jun Liu,† Nik Veldhoen,§ Cory Dubetz,‡ Caren C. Helbing,§ and John R. Cosgrove† †
AXYS Analytical Services Ltd. 2045 Mills Road West, Sidney, British Columbia V8L 5X2, Canada Institute of Ocean Sciences, Fisheries and Oceans Canada, 9860 West Saanich Road, Sidney, British Columbia V8L 4B2, Canada § Department of Biochemistry & Microbiology, University of Victoria, P.O. Box 1700 Stn CSC, Victoria, British Columbia V8W 2Y2, Canada ‡
S Supporting Information *
ABSTRACT: The health of Skeena River Sockeye salmon (Onchorhychus nerka) has been of increasing concern due to declining stock returns over the past decade. In the present work, in-migrating Sockeye from the 2008 run were evaluated using a mass spectrometry-based, targeted metabolomics platform. Our objectives were to (a) investigate natural changes in a subset of the hepatic metabolome arising from migration-associated changes in osmoregulation, locomotion, and gametogenesis, and (b) compare the resultant profiles with animals displaying altered hepatic vitellogenin A (vtg) expression at the spawning grounds, which was previously hypothesized as a marker of xenobiotic exposure. Of 203 metabolites monitored, 95 were consistently observed in Sockeye salmon livers and over half of these changed significantly during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health of migrating Skeena Sockeye.
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INTRODUCTION
km up the Skeena River to the Babine Lake spawning grounds and, over the course of migration, undergo dramatic physiological and morphological changes. Sex steroids (e.g., estradiol and testosterone), associated with stimulated gametogenesis, are responsible for dramatic alterations in the physical appearance of the salmon. These changes generally occur over a period of weeks to months and reach their peaks toward the end of in-migrations.5 A recent examination of hepatic gene expression profiles in Sockeye from the Fraser and Skeena Rivers revealed marked changes in sex-specific gene expression during the 2008 Skeena spawning migration.6 Among the changes was increased variability in the expression of mRNA encoding vitellogenin A (vtg), the main phospholipoglycoprotein used to transport lipids from storage sites to the oocytes during gonadal
The Skeena River watershed is the second largest freshwater habitat for Sockeye salmon (Onchorhychus nerka) in British Columbia (BC), Canada (Figure 1). Within this region, the spawning grounds of Fulton River and Pinkut Creek on Babine Lake account for the majority of total Skeena Sockeye production.1 Installation of spawning channels and flow controls at these sites in the 1970s produced a rapid increase in stock returns (catch plus escapement) up until the mid 1990s.2 A concomitant drop in returns and production began in the Babine Lake system in the early 2000s and has since continued unabated over the past decade, despite declines in exploitation.3 In August 2013, Fisheries and Oceans Canada placed a moratorium on fishing activities along the Skeena River, including its lakes and tributaries.4 The average Sockeye lifecycle is 4 years, consisting of one or more years in the ocean followed by a return to natal spawning grounds. Prior to in-migration, salmon cease feeding and rely solely on muscle and lipid stores as fuel for locomotion, gametogenesis, and osmoregulation. Sockeye migrate over 500 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 11670
July 5, 2014 September 3, 2014 September 8, 2014 September 8, 2014 dx.doi.org/10.1021/es503266x | Environ. Sci. Technol. 2014, 48, 11670−11678
Environmental Science & Technology
Article
molecular perturbation. Taken together, the results form a baseline of metabolite data against which future stock assessments can be compared for ongoing management of the health of Skeena River Sockeye.
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MATERIALS AND METHODS Metabolite Targets of Interest. A total of 203 metabolites were investigated, including 21 amino acids (AAs), 20 biogenic amines (BAs), 40 acylcarnitines (ACs), 89 phosphatidylcholines (PCs), 15 sphingomyelins (SMs), ∑hexose (Hex), and 17 fatty acids (FAs). This panel represents a diverse set of metabolites covering multiple systems (a subset of the entire “metabolome”) suitable for probing a variety of health end points in a wide range of species. A full list of analytes, internal standards, and abbreviations is provided in Tables S1−S4 of the Supporting Information (SI). Sampling. Sampling of adult Sockeye salmon took place during the 2008 spawning migration at the mouth of the Skeena River (July 29−30; n = 41 genotypic males, n = 35 genotypic females; all identified as Fulton stock), and spawning grounds in Pinkut Creek (September 11; n = 24 genotypic males and n = 26 genotypic females), and Fulton River (September 24; n = 23 genotypic males and n = 20 genotypic females) using capture by gill or seine nets. Fish were killed immediately following capture by a blow to the head; after which scales were removed for DNA fingerprint-based stock identification and liver sections were removed for analysis of mRNA transcript abundance. Remaining liver samples were placed in individual Whirl-pak bags on ice and then stored at −20 °C immediately upon return to the Skeena regional laboratory. The time between liver removal and placement of samples into freezers (2 h) was kept relatively constant to minimize handling-associated effects on metabolite levels across all sample collection sites. Following shipment to the Institute of Ocean Sciences, liver samples were stored at −50 °C prior to analysis. Genotypic and phenotypic anchoring. Stock assessment via DNA-based fingerprinting was conducted on all animals at the Pacific Biological Station Molecular Genetics Laboratory (Nanaimo, BC).12,13 Salmon from the mouth of the Skeena were confirmed as Fulton Stock. For animals collected at the spawning grounds, the results of DNA-based stock assessment matched the site of collection in all instances (i.e., animals sampled at Fulton were exclusively Fulton stock while animals sampled at Pinkut were exclusively Pinkut stock). Salmon were grouped according to genotypic, gametic, and molecular phenotypic sex using methods described in detail elsewhere.6 Briefly, genotypic sex was assessed via a quantitative real-time polymerase chain reaction (qPCR) assay using genomic DNA from each fish with amplification of the male-specific OTY2WSU locus as described in Veldhoen et al. (2010, 2013).6,14 Gametic sex was assessed during collections by visual confirmation of the presence of milt or roe. Molecular phenotypic sex was based on relative levels of qPCR-derived hepatic vtg mRNA abundance, with the female phenotype displaying vtg transcript levels of ≥100 and the male phenotype associated with values of
