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  • OPEN ACCESS
    Host–parasite transcriptomics during immunostimulant-enhanced rejection of salmon lice (Lepeophtheirus salmonis) by Atlantic salmon (Salmo salar)
    FACETS • Vol. 2 • pp. 477 - 495
    Salmon lice (Lepeophtheirus salmonis) are important ectoparasites of wild and farmed salmonids and cause major losses to the salmon farming industry throughout the Northern Hemisphere. With the emergence of resistance to several commonly used parasiticides, novel control strategies and integration of multiple treatment options are needed, including host immunostimulation. Here, we investigate the effects of a functional feed containing a peptidoglycan and nucleotide formulation on L. salmonis infection of Atlantic salmon (Salmo salar) by characterizing lice infection levels, the expression of several host immune genes, and the parasite transcriptomic response to the immunostimulated host. Although initial infection intensities were low, the low dose (LD) immunostimulant diet reduced the total lice burden by 50% relative to controls. Immunostimulant fed hosts up-regulated interleukin-1β in the skin and spleen. This gene has been implicated in successful responses of several salmonid species to salmon lice but is typically not observed in Atlantic salmon, suggesting a favorable influence on the immune response. Lice infecting LD immunostimulated salmon overexpressed genes putatively involved in parasite immunity, including carboxylesterases, and underexpressed genes putatively involved in feeding (e.g., proteases). These lice response genes further improve the characterization of the transcriptome of the non-model parasite by identifying genes potentially involved in evading host immunity.
  • OPEN ACCESS
    Prelude to a panzootic: Gene flow and immunogenetic variation in northern little brown myotis vulnerable to bat white-nose syndrome
    FACETS • Vol. 2 • pp. 690 - 714
    The fungus that causes bat white-nose syndrome (WNS) recently leaped from eastern North America to the Pacific Coast. The pathogen’s spread is associated with the genetic population structure of a host (Myotis lucifugus). To understand the fine-scale neutral and immunogenetic variation among northern populations of M. lucifugus, we sampled 1142 individuals across the species’ northern range. We used genotypes at 11 microsatellite loci to reveal the genetic structure of, and directional gene flow among, populations to predict the likely future spread of the pathogen in the northwest and to estimate effective population size (Ne). We also pyrosequenced the DRB1-like exon 2 of the class II major histocompatibility complex (MHC) in 160 individuals to explore immunogenetic selection by WNS. We identified three major neutral genetic clusters: Eastern, Montane Cordillera (and adjacent sampling areas), and Haida Gwaii, with admixture at intermediate areas and significant substructure west of the prairies. Estimates of Ne were unexpectedly low (289–16 000). Haida Gwaii may provide temporary refuge from WNS, but the western mountain ranges are not barriers to its dispersal in M. lucifugus and are unlikely to slow its spread. Our major histocompatibility complex (MHC) data suggest potential selection by WNS on the MHC, but gene duplication limited the immunogenetic analyses.
  • OPEN ACCESS
    Cryptic species diversity and reproductive isolation among sympatric lineages of Strongylocentrotus sea urchins in the northwest Atlantic
    FACETS • Vol. 3 • No. 1 • pp. 61 - 78
    Distinguishing between intra- and inter-specific variation in genetic studies is critical to understanding evolution because the mechanisms driving change among populations are expected to be different than those that shape reproductive isolation among lineages. Genetic studies of north Atlantic green sea urchins Strongylocentrotus droebachiensis (Müller, 1776) have detected significant population substructure and asymmetric gene flow from Europe to Atlantic Canada and interspecific hybridization between S. droebachiensis and Strongylocentrotus pallidus (Sars, 1871). However, combined with patterns of divergence at mtDNA sequences, morphological divergence at gamete traits suggests that the European and North American lineages of S. droebachiensis may be cryptic species. Here, we use a combination of cytochrome c oxidase subunit I (COI) sequences and single nucleotide polymorphisms (SNPs) to test for cryptic species within Strongylocentrotus sea urchins and hybrids between S. droebachiensis and S. pallidus populations. We detect striking patterns of habitat and reproductive isolation between two S. droebachiensis lineages, with offshore deep-water collections consisting of S. pallidus in addition to a cryptic lineage sharing genetic similarity with previously published sequences from eastern Atlantic S. droebachiensis. We detected only limited hybridization among all three lineages of sea urchins, suggesting that shared genetic differences previously reported may be a result of historical introgression or incomplete lineage sorting.
  • OPEN ACCESS
    Sex-biased gene expression and evolution of candidate reproductive transcripts in adult stages of salmon lice (Lepeophtheirus salmonis)
    FACETS • Vol. 4 • No. 1 • pp. 254 - 274
    The salmon louse Lepeophtheirus salmonis (Krøyer 1837) displays numerous sexually dimorphic characteristics. Insights into their underlying molecular components have only recently been explored, which serve to better understand both the basic biology of the louse, associated impacts on drug sensitivity, and evolution of resistance. Expression of 16 L. salmonis genes putatively involved in sexual dimorphism and reproduction were used to determine differences between sexes and better understand responses to mating using RT-qPCR of pre-adult and adult lice. Analysis of these genes revealed the dynamic nature of sex-biased expression across stages. However, female reception of a spermatophore did not appear to impact the expression of these particular genes. Furthermore six of these transcripts and 84 others identified previously in a large-scale louse transcriptomics experiment were used to estimate differences in evolutionary rates and codon-usage bias of sex-related genes using phylogenetic analysis by maximum likelihood (PAML) and codonW. Results suggest male-biased genes are evolving at significantly greater rates than female-biased and unbiased genes as evidenced by higher rates of non-synonymous substitutions and lower codon-usage bias in these genes. These analyses expand our understanding of interactions of sex-biased expression across the pre-adult and adult life stages and provide foundations for better understanding evolutionary differences in sex-biased genes of L. salmonis.
  • OPEN ACCESS
    A molecular assessment of infectious agents carried by Atlantic salmon at sea and in three eastern Canadian rivers, including aquaculture escapees and North American and European origin wild stocks
    FACETS • Vol. 5 • No. 1 • pp. 234 - 263
    Infectious agents are key components of animal ecology and drivers of host population dynamics. Knowledge of their diversity and transmission in the wild is necessary for the management and conservation of host species like Atlantic salmon (Salmo salar). Although pathogen exchange can occur throughout the salmon life cycle, evidence is lacking to support transmission during population mixing at sea or between farmed and wild salmon due to aquaculture exposure. We tested these hypotheses using a molecular approach that identified infectious agents and transmission potential among sub-adult Atlantic salmon at marine feeding areas and adults in three eastern Canadian rivers with varying aquaculture influence. We used high-throughput qPCR to quantify infection profiles and next generation sequencing to measure genomic variation among viral isolates. We identified 14 agents, including five not yet described as occurring in Eastern Canada. Phylogenetic analysis of piscine orthoreovirus showed homology between isolates from European and North American origin fish at sea, supporting the hypothesis of intercontinental transmission. We found no evidence to support aquaculture influence on wild adult infections, which varied relative to environmental conditions, life stage, and host origin. Our findings identify research opportunities regarding pathogen transmission and biological significance for wild Atlantic salmon populations.
  • OPEN ACCESS
    Comparing metabarcoding and morphological approaches to identify phytoplankton taxa associated with harmful algal blooms
    FACETS • Vol. 5 • No. 1 • pp. 784 - 811
    Molecular techniques are expected to be highly useful in detecting taxa causing harmful algal blooms (HABs). This is the first report in Canada evaluating HABs-related species identification using a combination of morphological and molecular approaches. Microscopy, quantitative polymerase chain reaction (qPCR), and metabarcoding with multiple markers (i.e., 16S, 18S-dinoflagellate and 18S-diatom, large subunit (28S) rDNA) were applied on samples (n = 54) containing suspected harmful algae (e.g., Alexandrium spp., Chattonella sp., Chrysochromulina spp., Dictyocha spp., Heterosigma akashiwo, Protoceratium reticulatum, Pseudochattonella verruculosa, Pseudo-nitzschia spp., Pseudopedinella sp.). Owing to methodology limitations, qPCR result interpretation was limited, although good detectability occurred using previously published assays for Alexandrium tamarense, H. akashiwo, and P. verruculosa. Overall, the multiple-marker metabarcoding results were superior to the morphology-based methods, with the exception of taxa from the silicoflagellate group. The combined results using both 18S markers and the 28S marker together closely corresponded with morphological identification of targeted species, providing the best overall taxonomic coverage and resolution. The most numerous unique taxa were identified using the 18S-dinoflagellate amplicon, and the best resolution to the species level occurred using the 28S amplicon. Molecular techniques are therefore promising for HABs taxa detection but currently depend on deploying multiple markers for metabarcoding.
  • OPEN ACCESS
    Phylogenomic characterization of ranaviruses isolated from cultured fish and amphibians in Thailand
    FACETS • Vol. 5 • No. 1 • pp. 963 - 979
    Ranaviruses are emerging pathogens associated with worldwide epizootics in farmed and wild ectothermic vertebrates. In this study, we determined the full genomes of eight ranaviruses isolated from marbled sleeper goby (Oxyeleotris marmorata), goldfish (Carassius auratus), guppy (Poecilia reticulata), tiger frog (Hoplobatrachus tigerinus), Asian grass frog (Fejervarya limnocharis), and East Asian bullfrog (H. rugulosus) cultured or imported into Thailand. These ranaviral isolates induced the same cytopathic effects (i.e., progression of coalescing round plaques) in epithelioma papulosum cyprini (EPC) cell cultures. Transmission electron microscopy of infected EPC cells revealed cytoplasmic viral particles with ultrastructural features typical for ranaviruses. Pairwise genetic comparisons of the complete major capsid protein coding sequences from the Thai ranaviruses displayed the highest identity (99.8%–100%) to a ranavirus (tiger frog virus; TFV) isolated from diseased tiger frogs cultured in China, a slightly lower identity (99.3%–99.4%) to a ranavirus (Wamena virus; WV) isolated from diseased green tree pythons (Morelia viridis) illegally exported from Papua New Guinea, and a lower identity to 35 other ranaviruses (93.7%–98.6%). Phylogenomic analyses supported the eight Thai ranaviruses, Chinese TFV, and WV as a subclade within a larger frog virus 3 clade. Our findings confirm the spread of TFV among cultured fish and amphibians in Asia and likely in reptiles in Oceania. Biosecurity measures are needed to ensure TFV does not continue to spread throughout Southeast Asia and to other parts of the world via international trade.
  • OPEN ACCESS
    Detection of carbapenem-resistance genes in bacteria isolated from wastewater in Ontario
    FACETS • Vol. 6 • pp. 569 - 591
    Bacterial carbapenem resistance is a major public health concern since these antimicrobials are often the last resort to treat serious human infections. To evaluate methodologies for detection of carbapenem resistance, carbapenem-tolerant bacteria were isolated from wastewater treatment plants in Toronto, Ottawa, and Arnprior, Ontario. A total of 135 carbapenem-tolerant bacteria were recovered. Polymerase chain reaction (PCR) indicated the presence of carbapenem hydrolysing enzymes KPC (n = 10), GES (n = 5), VIM (n = 7), and IMP (n = 1), and β-lactamases TEM (n = 7), PER (n = 1), and OXA-variants (n = 16). A subset of 46 isolates were sequenced and analysed using ResFinder and CARD-RGI. Both programs detected carbapenem resistance genes in 35 sequenced isolates and antimicrobial resistance genes (ARGs) conferring resistance to multiple class of other antibiotics. Where β-lactamase resistance genes were not initially identified, lowering the thresholds for ARG detection enabled identification of closely related β-lactamases. However, no known carbapenem resistance genes were found in seven sequenced Pseudomonas spp. isolates. Also of note was a multi-drug-resistant Klebsiella pneumoniae isolate from Ottawa, which harboured resistance to seven antimicrobial classes including β-lactams. These results highlight the diversity of genes encoding carbapenem resistance in Ontario and the utility of whole genome sequencing over PCR for ARG detection where resistance may result from an assortment of genes.
  • OPEN ACCESS
    Way out there: pathogens, health, and condition of overwintering salmon in the Gulf of Alaska
    FACETS • Vol. 7 • pp. 247 - 285
    Salmon are keystone species across the North Pacific, supporting ecosystems, commercial opportunities, and cultural identity. Nevertheless, many wild salmon stocks have experienced significant declines. Salmon restoration efforts focus on fresh and coastal waters, but little is known about the open ocean environment. Here we use high throughput RT-qPCR tools to provide the first report on the health, condition, and infection profile of coho, chum, pink, and sockeye salmon in the Gulf of Alaska during the 2019 winter. We found lower infectious agent number, diversity, and burden compared with coastal British Columbia in all species except coho, which exhibited elevated stock-specific infection profiles. We identified Loma sp. and Ichthyophonus hoferi as key pathogens, suggesting transmission in the open ocean. Reduced prey availability, potentially linked to change in ocean conditions due to an El Niño event, correlated with energetic deficits and immunosuppression in salmon. Immunosuppressed individuals showed higher relative infection burden and higher prevalence of opportunistic pathogens. We highlight the cumulative effects of infection and environmental stressors on overwintering salmon, establishing a baseline to document the impacts of a changing ocean on salmon.
  • OPEN ACCESS
    Amplicon-based and metagenomic approaches provide insights into toxigenic potential in understudied Atlantic Canadian lakes
    FACETS • Vol. 7 • pp. 194 - 214
    Cyanobacterial blooms and their toxigenic potential threaten freshwater resources worldwide. In Atlantic Canada, despite an increase of cyanobacterial blooms in the last decade, little is known about the toxigenic potential and the taxonomic affiliation of bloom-forming cyanobacteria. In this study, we employed polymerase chain reaction (PCR) and metagenomic approaches to assess the potential for cyanotoxin and other bioactive metabolite production in Harvey Lake (oligotrophic) and Washademoak Lake (mesotrophic) in New Brunswick, Canada, during summer and early fall months. The PCR survey detected the potential for microcystin (hepatotoxin) and anatoxin-a (neurotoxin) production in both lakes, despite a cyanobacterial bloom only being visible in Washademoak. Genus-specific PCR associated microcystin production potential with the presence of Microcystis in both lakes. The metagenomic strategy provided insight into temporal variations in the microbial communities of both lakes. It also permitted the recovery of a near-complete Microcystis aeruginosa genome with the genetic complement to produce microcystin and other bioactive metabolites such as piricyclamide, micropeptin/cyanopeptolin, and aeruginosin. Our approaches demonstrate the potential for production of a diverse complement of bioactive compounds and establish important baseline data for future studies of understudied lakes, which are frequently affected by cyanobacterial blooms.
  • OPEN ACCESS
    Association of CLOCK gene variants with obesity and adiposity-related anthropometric, metabolic, and behavioral parameters
    FACETS • Vol. 7 • pp. 792 - 808
    The CLOCK gene is a core component of the circadian clock and regulates various aspects of metabolism. Therefore, any variation that affects the function/expression of the CLOCK gene may contribute to the manifestation of metabolic disorders such as obesity. This study investigated whether the CLOCK variants rs4864548 and rs6843722 are associated with obesity and related traits in Pakistanis. A total of 306 overweight/obese cases and 306 age- and gender-matched control subjects were recruited (males 336 and females 276, age range 12–63 years). Anthropometric and metabolic parameters were taken by standard procedures and biochemical analyses, respectively. Behavior-related information was collected with a questionnaire. The genotypes of the variants were determined by allelic discrimination Taqman assays. Both variants were found to have a significant association with overweight/obesity according to the over-dominant model. The rs4864548 and rs6843722 were observed to escalate the risk of overweight/obesity by 1.611 (p = 0.004) and 1.657 (p = 0.002) times, respectively. These variants were also seen to be significantly associated with various other adiposity-related anthropometric parameters (p < 0.05). However, no association of both variants with metabolic and behavioral parameters was observed (p > 0.05). Thus, these variants may contribute to increasing the risk of overweight/obesity and related anthropometric traits in Pakistanis.
  • OPEN ACCESS
    Population structure of lake whitefish (Coregonus clupeaformis) from the Mississippian lineage in North America
    FACETS • Vol. 7 • pp. 853 - 874
    The lake whitefish (Coregonus clupeaformis) is a commercially valuable freshwater species with a broad distribution in North America. Some phylogeographic work has been done on this species, but little is known about genetic population subdivision among populations of the widely dispersed Mississippian lineage. We used 3,173 single nucleotide polymorphisms in 508 lake whitefish from 22 different lakes to examine population structure across central Canada and the United States. Bayesian clustering, ordination, and fixation indices identified population subdivision that largely reflected geographic distance and hydrological connectivity, with greater differentiation between lakes that are farther apart. Population subdivision was hierarchical, with greater differentiation between Canadian provinces and less differentiation based on river basins within provincial boundaries. Interestingly, isolation by distance alone was not sufficient to account for all of the observed genetic differentiation among populations. We conclude that important components of lake whitefish genetic diversity are present at different spatial scales, and that populations within the Mississippian lineage have differentiated widely across their range.
  • OPEN ACCESS
    Using transcriptomics to examine the physiological status of wild-caught walleye (Sander vitreus)
    FACETS • Vol. 8 • pp. 1 - 15
    Lake Winnipeg, Manitoba, is Canada’s second largest commercial inland freshwater fishery, and concern over collapse of the walleye fishery is growing. Molecular techniques have been increasingly used to study responses of wild organisms to environmental and anthropogenic stressors. The present study used transcriptomics to examine the physiology of wild-caught walleye (Sander vitreus (Mitchell, 1818)) across Lake Winnipeg using non-lethal techniques. Gill transcriptomes of walleye sampled from the north and south basins of Lake Winnipeg, and the channel connecting them, exhibited distinct profiles implicating regionally specific biological responses. North basin walleye exhibited transcriptomic responses indicative of exposure to environmental stressors. Transcriptomic patterns suggested a shift to increased reliance on anaerobic metabolism and up-regulation of hypoxia-sensitive genes in north basin fish, possibly representing exposure to low-oxygen conditions. Exposure to environmental stressors may also have driven increases in gene transcripts associated with proteasomal catabolism, DNA repair, molecular chaperones, and ion regulation. North basin fish also exhibited transcriptomic patterns indicative of gill remodeling via regulation of the mTOR pathway, cytoskeleton reorganization, and fatty acid synthesis. Our results highlight the complexity of examining wild fish across environmental gradients but also the potential use of molecular techniques in elucidating organismal sensitivity to local environmental factors.
  • OPEN ACCESS
    Still using genetic data? A comparative review of Canadian life insurance application forms before and after the GNDA
    FACETS • Vol. 9 • pp. 1 - 10
    Genetic testing's increased availability has raised concerns about “genetic discrimination” (GD), where individuals may face unfair treatment, particularly when purchasing personal insurance, because of their genetic characteristics. In 2017, Canada passed the Genetic Non-Discrimination Act (GNDA) to prevent GD. This manuscript reviews post-GNDA life insurance application forms and compares them with pre-2014 application forms to assess the impact of the GNDA on insurance practices in Canada. Based on our comparative assessment, we found that the GNDA has had a modest impact on the practice of life insurers in Canada. Our study also confirms that questions about family history of disease and broadly formulated inquiries are still used on life insurance application forms. Both can, in the absence of clear instructions, lead applicants to disclose protected genetic information.
  • OPEN ACCESS
    Marine ecozones represent a major gap in Canadian DNA barcoding efforts
    FACETS • Vol. 9 • pp. 1 - 11
    Because of Canada’s large size, it is impractical to obtain a comprehensive perspective on biotic change through morphological approaches. DNA metabarcoding offers a potential path, but its application requires access to a well-parameterized DNA barcode reference library. This study presents the current state of DNA barcode coverage for Canadian animals, highlighting progress, identifying gaps, and providing recommendations for future research. Our analysis indicates that many of the known species (100 000 terrestrial and 6000 marine) in the Canadian fauna possess DNA barcode coverage, but there are important gaps geographically and taxonomically. We summarize DNA barcode coverage for the species in freshwater, marine, and terrestrial environments by ecoregion, finding that 95.6% of the 2.3 million Canadian barcode records derive from terrestrial ecosystems. Although the density of barcode records per 100 km² is 13x higher for terrestrial than aquatic environments (22.4 vs. 1.7), coverage for 58% of marine species is available (54% for annelids, 52% for arthropods, 88% for chordates, 39% for echinoderms, and 46% for molluscs). By revealing data-deficient areas and taxonomic groups, this study offers a roadmap for expanding the DNA barcode library for the Canadian fauna as an essential foundation for the scalable biosurveillance initiatives that inform biodiversity conservation efforts.
  • OPEN ACCESS
    American lobster (Homarus americanus) hepatopancreas transcriptome reveals the significance of chitin-related genes during impoundment shell disease
    FACETS • Vol. 9 • pp. 1 - 10
    American lobsters (Homarus americanus) stored in open tidal pounds can develop impoundment shell disease (ISD), resulting in decreased marketability of the lobsters on the live market. Little is known about ISD or the immunological responses of lobsters exhibiting this disease. The objective of this project was to identify genes from H. americanus hepatopancreas that are differentially expressed in response to ISD. Lobsters were separated into asymptomatic, moderately symptomatic, and severely symptomatic groups, which represent animals with 0%, 5%–20%, and >20% lesion coverage of the carapace, respectively. RNA-seq analysis found that 134 genes were differentially expressed between groups (false discovery rate (FDR) < 0.05). Most, 80, of these genes were found exclusively in the comparison between moderately symptomatic and asymptomatic groups. All animals clustered in their proposed groups based on the expression of the differently expressed genes (DEGs), and the asymptomatic group clustered as an out-group. The expression of most DEGs was higher in the asymptomatic group than the others, which could be related to a stronger response against the disease or differences in individual resistance against ISD development. Among these genes, we highlight eight chitin-related genes, one α-2-macroglobulin-like gene, one acute phase serum amyloid A gene, one pseudohaemocyanin gene, and one trypsin-1-like gene.