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In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment.

ABSTRACT: Genetic stock identification (GSI) from genotyping-by-sequencing of single nucleotide polymorphism (SNP) loci has become the gold standard for stock of origin identification in Pacific salmon. The sequencing platforms currently applied require large batch sizes and multiday processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, such as the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and promise real-time, in-field stock identification of variable batch sizes. Here we evaluate utility and comparability to established GSI platforms of at-sea stock identification of coho salmon (Oncorhynchus kisutch) using targeted SNP amplicon sequencing on the minION platform during a high-sea winter expedition to the Gulf of Alaska. As long read sequencers are not optimized for short amplicons, we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded data sufficient for stock assignment for 50 out of 80 individuals. Nanopore-based SNP calls agreed with Ion Torrent-based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals, highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed salmon in the queried baseline data set contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time and cost, increasing accuracy and throughput, as well as augmentation of the existing baselines. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach by providing mobile small batch genotyping to diverse stakeholders.

SUBMITTER: Deeg CM

PROVIDER: S-EPMC9303916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Wild Pacific salmon, including Coho salmon Onchorynchus kisutch, have been supplemented with hatchery propagation for over 50 years in support of increased ocean harvest and conservation of threatened populations. In Canada, the Wild Salmon Policy for Pacific salmon was established with the goal of maintaining and restoring healthy and diverse Pacific salmon populations, making conservation of wild salmon and their habitats the highest priority for resource management decision-making. A new approach to the assessment and management of wild coho salmon, and the associated hatchery production and fishery management is needed. Implementation of parentage-based tagging (PBT) may overcome problems associated with coded-wire tag-based (CWT) assessment and management of coho salmon fisheries, providing at a minimum information equivalent to that derived from the CWT program. PBT and genetic stock identification (GSI) were used to identify coho salmon sampled in fisheries (8,006 individuals) and escapements (1,692 individuals) in British Columbia to specific conservation units (CU), populations, and broodyears. Individuals were genotyped at 304 single nucleotide polymorphisms (SNPs) via direct sequencing of amplicons. Very high accuracy of assignment to population (100%) via PBT for 543 jack (age 2) assigned to correct age and collection location and 265 coded-wire tag (CWT, age 3) coho salmon assigned to correct age and release location was observed, with a 40,774-individual, 267-population baseline available for assignment. Coho salmon from un-CWTed enhanced populations contributed 65% of the catch in southern recreational fisheries in 2017. Application of a PBT-GSI system of identification to individuals in 2017 fisheries and escapements provided high-resolution estimates of stock composition, catch, and exploitation rate by CU or population, providing an alternate and more effective method in the assessment and management of Canadian-origin coho salmon relative to CWTs, and an opportunity for a genetic-based system to replace the current CWT system for coho salmon assessment.

Project description:Coho salmon are a critical Pacific salmon species that undergo complex physiological transformations as they migrate towards seawater and enter adult life stages. During these periods, coho may receive exposure to waterborne pollutants that coincide with outmigration through contaminated waterways and return to natal streams. However, little is known regarding the ontogenic modulation of gene expression during these critical life stages. Accordingly, the purpose of the present study was to characterize the hepatic transcriptome of smolting coho, adult males, and adult females by carrying out microarray analysis with a commercially available 16,000 cDNA element platform. Quantitative PCR (Q-PCR) analysis of genes involved in chemical biotransformation (cytochrome P450 isoforms 1A, and 2M1, glutathione S-transferase pi, microsomal GST), defense against metal exposure (metallothionein-A), and reproductive function (vitellogenin receptor) were developed for the purpose of analyzing specific genes of interest and to validate the microarray data. Microarray analysis identified 842 genes that were differentially expressed between smolts and adult males or females (p<0.001 and more than 2-fold difference). These 842 genes were not differentially expressed between adult males and females and, therefore, can be interpreted as a smolt-specific transcriptional profile. Of these 842 genes, 275 were well annotated and formed the basis for further bioinformatics analysis. Many of the differentially expressed genes were involved in basic cellular processes related to protein biosynthesis and degradation (24%), ion transport (12%), transcription (8%), cell structure (8%) and cellular energetics (6%). The majority of differentially expressed genes involved in signal transduction and energy metabolism were expressed at higher levels in adult coho relative to smolts. However, genes associated with cellular protection against chemical injury (i.e. biotransformation, DNA damage repair, and protection against oxidative stress) did not generally differ among the groups. Q-PCR studies revealed extensive interindividual variation in mRNA expression, but were consistent with the microarray results (R(2)=0.74). Collectively, our results indicate differences in liver gene expression in young smolting coho salmon relative to adults and extensive interindividual variation in biotransformation gene expression. However, we did not find a global lack of hepatic biotransformation capacity or poor cellular detoxification response capacity in smolting cohos based on mRNA profiles.

Project description:Wild stocks of Pacific salmon in the Northwestern United States have declined in recent years, and the major factors contributing to these losses include water pollution and loss of habitat. In salmon, sublethal chemical exposures may impact critical behaviors (such as homing, feeding, predator-avoidance) that are important for species survival. Therefore, understanding the potential for these species to biotransform organic compounds within sensitive target tissues such as liver, gills and olfactory region can help estimate or predict their susceptibility to pollutants. In this study, we used real-time quantitative polymerase chain reaction (Q-PCR), Western blotting, and catalytic assays to characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch), a sensitive species in the Pacific Northwest. Gene expression analysis using Q-PCR assays developed for coho genes revealed the presence of the predominant cytochrome P450 mRNAs (CYP1A, CYP2K1, CYP2M1, CYP3A27) in the olfactory rosettes and provided quantitative mRNA expression levels in coho liver and gills. Q-PCR analysis revealed relatively high expression of the major CYP isoforms in the liver and olfactory rosettes, which was generally confirmed by Western blotting. Extrahepatic CYP expression was generally higher in the olfactory rosettes as compared to the gills. Catalytic studies demonstrated functional CYP1A-dependent ethoxyresorufin-O-deethylase, CYP2-dependent pentoxyresorufin-O-dealkylase, CYP2K1-dependent testosterone 16beta-hydroxylase, and CYP3A27-dependent testosterone 6beta-hydroxylase activities in liver, but not at detectable levels in gills. In contrast, flavin-containing monooxygenase (FMO)-dependent thiourea S-oxidase activity was readily observed in the gills and was substantially higher than that observed in liver. Collectively, the results of this study suggest that the olfactory rosettes are important sites of extrahepatic biotransformation in coho salmon, and that tissue specific-differences in Phase I metabolism may lead to contrasting tissue-specific biotransformation capabilities in this species.

Project description:The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

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In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment.

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