[Show abstract][Hide abstract] ABSTRACT: Disease outbreaks with the salmon louse Lepeophtheirus salmonis cause significant economic losses in mariculture operations worldwide. Variable innate immune responses at the louse-attachment site con-tribute to differences in susceptibility among species such that members of Salmo spp. are more susceptible to infection than those of some Oncorhynchus spp. Relatively little is known about the mechanisms that contribute to disease resistance or susceptibility to L. salmonis in salmon. Here, we utilize histochemis-try and transcriptomics in a comparative infection model with susceptible (Atlantic, sockeye) and resistant (coho) salmon. At least three cell populations (MHIIβ+, IL1β+, TNFα+) were activated in coho salmon skin during L. salmonis infection. Locally elevated expression of several pro-inflammatory mediators (e.g. IL1β, IL8, TNFα, COX2, C/EBPβ), and tissue repair enzymes (MMP9, MMP13) were detected in susceptible and resistant species. However, responses specific to coho salmon (e.g. IL4, IL6, TGFβ) or responses shared among susceptible salmon (e.g. SAP, TRF, Cath in Atlantic and sockeye salmon) provide evidence for species-specific pathways contributing to resistance or susceptibility, respectively. Our results confirm the importance of an early pro-inflammatory TH1-type pathway as an initial host response during infection with Pacific sea lice, and demonstrate subsequent regulatory TH2-type processes as candidate defense mechanisms in the skin of resistant coho salmon.
[Show abstract][Hide abstract] ABSTRACT: Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs that might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals.
Developmental and comparative immunology. 08/2014;
[Show abstract][Hide abstract] ABSTRACT: The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the Atlantic salmon and rainbow trout genomes by facilitating their comparison with other major teleost groups. Citation: Rondeau EB, Minkley DR, Leong JS, Messmer AM, Jantzen JR, et al. (2014) The Genome and Linkage Map of the Northern Pike (Esox lucius): Conserved Synteny Revealed between the Salmonid Sister Group and the Neoteleostei. PLoS ONE 9(7): e102089. doi:10.1371/journal.pone.0102089
PLoS ONE 07/2014; 9(7):e102089. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salmon species vary in susceptibility to infections with the salmon louse (Lepeophtheirus salmonis). Comparing mechanisms underlying responses in susceptible and resistant species is important for estimating impacts of infections on wild salmon, selective breeding of farmed salmon, and expanding our knowledge of fish immune responses to ectoparasites. Herein we report three L. salmonis experimental infection trials of co-habited Atlantic Salmo salar, chum Oncorhynchus keta and pink salmon O. gorbuscha, profiling hematocrit, blood cortisol concentrations, and transcriptomic responses of the anterior kidney and skin to the infection.
In all trials, infection densities (lice per host weight (g)) were consistently highest on chum salmon, followed by Atlantic salmon, and lowest in pink salmon. At 43 days post-exposure, all lice had developed to motile stages, and infection density was uniformly low among species. Hematocrit was reduced in infected Atlantic and chum salmon, and cortisol was elevated in infected chum salmon. Systemic transcriptomic responses were profiled in all species and large differences in response functions were identified between Atlantic and Pacific (chum and pink) salmon. Pink and chum salmon up-regulated acute phase response genes, including complement and coagulation components, and down-regulated antiviral immune genes. The pink salmon response involved the largest and most diverse iron sequestration and homeostasis mechanisms. Pattern recognition receptors were up-regulated in all species but the active components were often species-specific. C-type lectin domain family 4 member M and acidic mammalian chitinase were specifically up-regulated in the resistant pink salmon.
Experimental exposures consistently indicated increased susceptibility in chum and Atlantic salmon, and resistance in pink salmon, with differences in infection density occurring within the first three days of infection. Transcriptomic analysis suggested candidate resistance functions including local inflammation with cytokines, specific innate pattern recognition receptors, and iron homeostasis. Suppressed antiviral immunity in both susceptible and resistant species indicates the importance of future work investigating co-infections of viral pathogens and lice.
[Show abstract][Hide abstract] ABSTRACT: Transposable elements (TEs) are DNA sequences capable of mutagenic proliferation within a genome and are exceptionally important to the molecular evolution of their host. Using a variety of complementary bioinformatics approaches we have characterized the distribution of TEs in the genome of a salmonid, Atlantic salmon (Salmo salar). Salmonids are a rapidly evolving species group whose common ancestor experienced a whole-genome duplication (WGD) event approximately 95 million years ago. WGDs have occurred only rarely in the vertebrate lineage and are followed by increases in genetic diversity, novel gene function, gene subfunctionalization and increased regulatory complexity; the processes involved in restoring genome stability following a WGD are only partially understood. We compared TE content in the genome of Atlantic salmon to that in the closest related lineage which did not experience a WGD, Esociformes, represented by northern pike (Esox lucius). The TE-derived component of the Atlantic salmon genome is substantially larger than the 20% we established for northern pike, indicating a massive increase in TE activity since the divergence of these two lineages. Further, Atlantic salmon has the highest genomic TE content of all fish -derived portion of the Atlantic salmon genome is substantially larger than that in the non-WGD sister taxa, Esociformes (20% TE-derived content in representative Esox lucius), and is the largest TE abundance of any fish genome sequenced to date. Using bioinformatics pipelines, BLAST and phylogenetic analyses we have identified at least 40 distinct families of Tc1-Mariner-like TEs in the genome of Atlantic salmon. By examining abundance, specific insertion sites and the sequence similarity between family members we have established periods of transposition activity for these TEs and have shown that the salmonid ancestor experienced a massive proliferation of Tc1-Mariner elements following the WGD; this proliferation continued during salmonid speciation and has contributed hundreds of millions of base-pairs to the genome. Many of these TEs appear to have first entered the genome through horizontal transfer from an unknown vector. This work provides insight into key processes involved in genomic evolution and has helped to improve the quality and utility of the Atlantic salmon genome sequence.
The authors wish to acknowledge the support of Compute Canada, the Natural Sciences and Engineering Research Council of Canada and the Province of British Columbia.
Mobile Genetic Elements and Genome Evolution, Santa Fe, USA; 03/2014
[Show abstract][Hide abstract] ABSTRACT: Divergent life history strategies occur in steelhead or rainbow trout Oncorhynchus mykiss, and many populations produce both migrant (anadromous fish that move to the ocean after rearing) and resident (do not migrate and remain in fresh water) individuals. Mechanisms leading to each type are only partially understood; while the general tendency of a population is heritable, individual tendency may be plastic, influenced by local environment. Steelhead hatchery programs aim to mitigate losses in wild stocks by producing trout that will migrate to the ocean and not compete with wild trout for limited freshwater resources. To increase our understanding of gill function in these migratory or resident phenotypes, here we compare gill transcriptome profiles of hatchery-released fish either at the release site (residents) or five river kilometers downstream while still in full fresh water (migrants). To test if any of these genes can be used as predictive markers for smoltification, we compared these genes between migrant-like and undifferentiated trout while still in the hatchery in a common environment (pre-release). Results confirmed the gradual process of smoltification, and the importance of energetics, gill remodeling, and ion transport capacity for migrants. Additionally, residents overexpressed transcripts involved in antiviral defenses, potentially for immune surveillance via dendritic cells in the gills. The best smoltification marker candidate was protein s100a4, expression of which was highly correlated with Na+, K+ ATPase (NKA) activity and smolt-like morphology in pre- and post-release trout gills.
[Show abstract][Hide abstract] ABSTRACT: Transposable elements (TEs) are DNA sequences capable of mutagenic proliferation in their host genome and are found in virtually all eukaryotes. We have recently shown that TEs occupy at least 55% of the genome of Atlantic salmon (Salmo salar). The salmonids are a rapidly evolving species group whose common ancestor underwent a whole-genome duplication (WGD) event between 25 and 100 MYA. WGDs have occurred only rarely in the vertebrate lineage and are followed by increases in genetic diversity, novel gene function, gene subfunctionalization and speciation; the processes involved in restoring genome stability following WGD are only partially understood. Using bioinformatics pipelines, BLAST and phylogenetic analyses we have identified 40 families of Tc1- Mariner-type TEs in the genome of Atlantic salmon. By examining TE insertion sites and the sequence similarity between family members we have established periods of TE activity and shown that the salmonid ancestor experienced a massive proliferation of Tc1- Mariner elements immediately following the WGD. Many of these TEs appear to have first entered the genome through horizontal transfer from an unknown vector. This work provides insight into key processes involved in genomic evolution and will improve the quality and utility of the Atlantic salmon genome sequence.
Pacific Ecology and Evolution Conference (PEEC) 2014, Bamfield, Canada; 02/2014
[Show abstract][Hide abstract] ABSTRACT: Inherited symbionts are ubiquitous in insects and can have important consequences for the fitness of their hosts. Many inherited symbionts defend their hosts against parasites or other natural enemies; however, the means by which most symbionts confer protection is virtually unknown. We examine mechanisms of defense in a recently discovered case of symbiont-mediated protection, where the bacterial symbiont Spiroplasma defends the fruit fly Drosophila neotestacea from a virulent nematode parasite, Howardula aoronymphium. Using quantitative PCR of Spiroplasma infection intensities and whole transcriptome sequencing, we attempt to distinguish between the following modes of defense: symbiont-parasite competition, host immune priming, and the production of toxic factors by Spiroplasma. Our findings do not support a model of exploitative competition between Howardula and Spiroplasma to mediate defense, nor do we find strong support for host immune priming during Spiroplasma infection. Interestingly, we recovered sequence for putative toxins encoded by Spiroplasma, including a novel putative ribosome inactivating protein, transcripts of which are up-regulated in response to nematode exposure. Protection via the production of toxins may be a widely used and important mechanism in heritable defensive symbioses in insects. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Classical major histocompatibility complex (MHC) class II molecules play an essential role in presenting peptide antigens to CD4+ T lymphocytes in the acquired immune system. The non-classical class II DM molecule, HLA-DM in the case of humans, possesses critical function in assisting the classical MHC class II molecules for proper peptide loading and is highly conserved in tetrapod species. Although the absence of DM-like genes in teleost fish has been speculated based on the results of homology searches, it has not been definitively clear whether the DM system is truly specific for tetrapods or not. To obtain a clear answer, we comprehensively searched class II genes in representative teleost fish genomes and analyzed those genes regarding the critical functional features required for the DM system.
We discovered a novel ancient class II group (DE) in teleost fish and classified teleost fish class II genes into three major groups (DA, DB and DE). Based on several criteria, we investigated the classical/non-classical nature of various class II genes and showed that only one of three groups (DA) exhibits classical-type characteristics. Analyses of predicted class II molecules revealed that the critical tryptophan residue required for a classical class II molecule in the DM system could be found only in some non-classical but not in classical-type class II molecules of teleost fish.
Teleost fish, a major group of vertebrates, do not possess the DM system for the classical class II peptide-loading and this sophisticated system has specially evolved in the tetrapod lineage.
[Show abstract][Hide abstract] ABSTRACT: The products of dax1, foxl2a and mis have each been shown to have proliferative and/or differentiative activities during mammalian organogenesis. These factors also play a role in regulating the biosynthesis of estrogen, particularly by modulating the activity of aromatase cyp19a. We demonstrate the transcription and translation of these genes during salmon embryogenesis. We were able to track sex-specific differences in these processes through accurate determination of the sex of each embryo and larva examined from genotyped microsatellites. We detected sex- and stage-specific immunolabeling of the embryonic gut, kidney, gonads, neural cord and skeletal muscle by DAX-1, FOXL2A and MIS. These results indicate the potential of these factors to mediate proliferation and/or differentiation programs during development of these tissues. As well, immunolabeling of skeletal muscle by CYP19B1 throughout the study reveals probable neurogenic activity associated with peripheral radial glial cells and the growing embryonic musculature.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 11/2013; · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The sablefish (order: Scorpaeniformes) is an economically important species in commercial fisheries of the North Pacific and an emerging species in aquaculture. Aside from a handful of sequences in NCBI and a few published microsatellite markers, little is known about the genetics of this species. The development of genetic tools, including polymorphic markers and a linkage map will allow for the successful development of future broodstock and mapping of phenotypes of interest. The significant sexual dimorphism between females and males makes a genetic test for early identification of sex desirable.
A full mitochondrial genome is presented and the resulting phylogenetic analysis verifies the placement of the sablefish within the Scorpaeniformes. Nearly 35,000 assembled transcript sequences are used to identify genes and obtain polymorphic SNP and microsatellite markers. 360 transcribed polymorphic loci from two sablefish families produce a map of 24 linkage groups. The sex phenotype maps to sablefish LG14 of the male map. We show significant conserved synteny and conservation of gene-order between the threespine stickleback Gasterosteus aculeatus and sablefish. An additional 1843 polymorphic SNP markers are identified through next-generation sequencing techniques. Sex-specific markers and sequence insertions are identified immediately upstream of the gene gonadal-soma derived factor (gsdf), the master-sex determinant locus in the medaka species Oyzias luzonensis.
The first genomic resources for sablefish provide a foundation for further studies. Over 35,000 transcripts are presented, and the genetic map represents, as far as we can determine, the first linkage map for a member of the Scorpaeniformes. The observed level of conserved synteny and comparative mapping will allow the use of the stickleback genome in future genetic studies on sablefish and other related fish, particularly as a guide to whole-genome assembly. The identification of sex-specific insertions immediately upstream of a known master sex determinant implicates gsdf as an excellent candidate for the master sex determinant for sablefish.
[Show abstract][Hide abstract] ABSTRACT: We report sets of 19 and 16 microsatellite loci for the examination of the population genetics of Boreogadus saida and Arctogadus glacialis, respectively. Six of these loci were developed from a collection of 9,497 expressed sequences from B. saida while the remaining loci were found in the literature and optimized for use in B. saida and A. glacialis. The numbers of alleles observed for each locus ranged from 3 to 33 in B. saida and 1–22 in A. glacialis. Observed heterozygosities ranged from 0.02 to 0.93 in B. saida and 0.17–1.0 in A. glacialis. Species specific differences were observed for the loci providing new tools for the identification of these two morphologically similar arctic gadids. The loci presented here can be used to distinguish between the two species and fill fundamental biological knowledge gaps, thus promoting conservation of these important fishes.
[Show abstract][Hide abstract] ABSTRACT: Transcripts for dax1, foxl2, mis and sf1 are co-expressed in the somatic companion cells of teleost germ cells. These regulatory factors function, in part, to modulate the transcription of aromatase, particularly cyp19a, the terminal enzyme of estrogen biosynthesis. At least two separate aromatase loci exist in teleost fish that encode distinct isoforms. The activity of two forms, cyp19a and cyp19b1, are predominantly associated with the ovary and the brain, respectively. We isolated sequences that compose the proximal promoters of cyp19a, cyp19b1 and foxl2a, to identify potential transcription factor binding motifs to define sex-specific regulatory profiles for each gene. We also provide evidence for the translation and immunological localization of DAX-1, FOXL2 and MIS to the endoplasmic reticulum and accumulation within secretory vesicles of the salmon oocyte. We found no evidence for the expression of CYP19A or CYP19B1 in the oocyte at the one-year-old stage. However, synthesis of both aromatases was localized to testicular germ and soma cells at this early stage of development. Production of these regulatory factors in the germ cells may serve to modulate the transcription and activity of endogenous aromatase and/or contribute to the differentiation of the neighbouring companion cells through secretory signaling.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 02/2013; · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular genetic tools have become standard in biological studies of both model and non-model species. This has created a growing need for sequence information, a resource hitherto limited for many species. With new sequencing technologies this is rapidly changing, and whole genome shotgun sequencing has become a realistic goal for many species. However, present sequencing protocols require more DNA than can be extracted from single individuals of many small metazoans, potentially forcing sequencing projects to perform sequencing on samples derived from several individuals. A pertinent question thus arises: can wild samples be used or is inbreeding necessary? In the present study we compare assemblies generated using sequence data from inbred and wild Lepeophtheirus salmonis. The results indicate not only that measures to reduce the genetic variability may significantly improve the final assemblies but also that deeper coverage to some extent can compensate for the detrimental effects of natural sequence variability.
Journal of Natural History 01/2013; 47(5-12):901-910. · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant products in general and soybeans in particular can challenge the function and health of the intestinal tract. Salmonids develop an intestinal inflammation when fed diets containing soybean meal (SBM) and certain other legume ingredients. In the present study a 44K oligonucleotide salmonid microarray, qPCR and histology were used to investigate early response mechanisms in the distal intestine of Atlantic salmon (Salmo salar L.) during the first week of oral exposure to a diet containing 20% extracted SBM. The distal intestine transcriptome was profiled on days 1, 2, 3, 5 and 7 and compared to a control group fed fishmeal as the sole protein source. Histological evaluation of the distal intestine revealed the first signs of inflamation on day 5. The most prominent gene expression changes were seen on days 3 and 5. Up-regulation in immune-related genes was observed during the first 5 days, including GTPase IMAP family members, NF-kB-related genes and regulators of T cell and B cell function. Many functional genes involved in lipid metabolism, proteolysis, transport, metabolism and detoxification were initially up-regulated on days 1-3, possibly as an attempt by the tissue to compensate for the initiating immune response. Cell repair and extracellular matrix remodeling genes were up-regulated (heparanase, collagenase) on days 3 and 5. Down regulation of genes related to endocytosis, exocytosis, detoxification, transporters and metabolic processes from day 5 indicated initiation of dysfuntion of digestive and metabolic functions that may occur as a result of inflammation or as a response to the introduction of soybean meal in the diet. This is the first study conducting transcriptomic profiling to characterize early responses during the development of SBMIE. Switching Atlantic salmon from a fishmeal to a 20% SBM diet resulted in rapid changes to the intestinal transcriptome, indicating an immune reaction with subsequent impaired epithelial barrier function and other vital intestinal functions.
Fish & Shellfish Immunology 12/2012; · 2.96 Impact Factor