Salmon sperm DNA increases sample recovery from cotton swabs.

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001-1-10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

Modeling ocean distributions and abundances of natural- and hatchery-origin Chinook salmon stocks with integrated genetic and tagging data.

Background: Considerable resources are spent to track fish movement in marine environments, often with the intent of estimating behavior, distribution, and abundance. Resulting data from these monitoring efforts, including tagging studies and genetic sampling, often can be siloed. For Pacific salmon in the Northeast Pacific Ocean, predominant data sources for fish monitoring are coded wire tags (CWTs) and genetic stock identification (GSI). Despite their complementary strengths and weaknesses in coverage and information content, the two data streams rarely have been integrated to inform Pacific salmon biology and management. Joint, or integrated, models can combine and contextualize multiple data sources in a single statistical framework to produce more robust estimates of fish populations. Methods: We introduce and fit a comprehensive joint model that integrates data from CWT recoveries and GSI sampling to inform the marine life history of Chinook salmon stocks at spatial and temporal scales relevant to ongoing fisheries management efforts. In a departure from similar models based primarily on CWT recoveries, modeled stocks in the new framework encompass both hatchery- and natural-origin fish. We specifically model the spatial distribution and marine abundance of four distinct stocks with spawning locations in California and southern Oregon, one of which is listed under the U.S. Endangered Species Act. Results: Using the joint model, we generated the most comprehensive estimates of marine distribution to date for all modeled Chinook salmon stocks, including historically data poor and low abundance stocks. Estimated marine distributions from the joint model were broadly similar to estimates from a simpler, CWT-only model but did suggest some differences in distribution in select seasons. Model output also included novel stock-, year-, and season-specific estimates of marine abundance. We observed and partially addressed several challenges in model convergence with the use of supplemental data sources and model constraints; similar difficulties are not unexpected with integrated modeling. We identify several options for improved data collection that could address issues in convergence and increase confidence in model estimates of abundance. We expect these model advances and results provide management-relevant biological insights, with the potential to inform future mixed-stock fisheries management efforts, as well as a foundation for more expansive and comprehensive analyses to follow.

ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Tidal gradients, fine-scale homing and a potential cryptic ecotype of wild spawning pink salmon (Oncorhynchus gorbuscha).

The homing behaviour of salmon is a remarkable natural phenomenon, critical for shaping the ecology and evolution of populations yet the spatial scale at which it occurs is poorly understood. This study investigated the spatial scale and mechanisms driving homing as depicted by spawning site-choice behaviour in pink salmon (Oncorhynchus gorbuscha) in Prince William Sound, Alaska. Molecular pedigree analyses of over 30,000 adult spawners in four streams revealed that pink salmon exhibit fine-scale site fidelity within a stream, returning to within <100 m of their parents. Homing behaviours were driven in part by a salinity gradient between intertidal and freshwater environments, with individuals incubated in freshwater environments more than twice as likely to spawn upstream of tidal influence than those incubated in the intertidal. Our findings challenge the traditional view that pink salmon populations are genetically and phenotypically homogenous due to their short freshwater residency as juveniles and high rates of dispersal as returning adults (i.e. straying). This study has important implications for rates of inbreeding, local adaptation and gene flow within populations, and is particularly relevant to the management of salmon hatcheries, given the high incidence of hatchery-origin pink salmon, reared in freshwater hatchery environments, that stray into wild populations of Prince William Sound.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Background: Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. Methods: By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. Results: We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Conclusion: Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

Diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) have altered microRNA responses in immune tissues after infection with Vibrio anguillarum.

Production of sterile fishes through artificial retention of a third set of chromosomes (triploidy) is a sustainable alternative for aquaculture since it reduces escapee pressure on wild populations. However, these fishes have reduced survival in stressful conditions and in response to infection. In this study, the impact of Vibrio anguillarum infection on diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut, head kidney, and spleen were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. All three tissues had differentially expressed miRNA prior to infection, indicating subtle changes in epigenetic regulation due to increased ploidy. Additionally, miRNA were altered by infection, but there was only a difference in spleen miRNA expression between diploids and triploids at three days of infection. Furthermore, one miRNA (ssa-miR-2188-3p) was confirmed as having an altered response to infection in triploids compared to diploids, implicating potential immune dysregulation due to increased ploidy. The miRNAs identified in this study are predicted to target immune pathways, providing evidence for their importance in regulating responses to pathogens. This study is the first to investigate how increased ploidy alters miRNA expression in response to infection. Additionally, it provides evidence for epigenetic dysregulation in triploid fishes, which may contribute to their poor performance in response to stress.

Regulation of host gene expression by gastrointestinal tract microbiota in Chinook Salmon (Oncorhynchus tshawytscha).

Differences in gut microbiome composition are linked with health, disease and ultimately host fitness; however, the molecular mechanisms underlying that relationship are not well characterized. Here, we modified the fish gut microbiota using antibiotic and probiotic feed treatments to address the effect of host microbiome on gene expression patterns. Chinook salmon (Oncorhynchus tshawytscha) gut gene expression was evaluated using whole transcriptome sequencing (RNA-Seq) on hindgut mucosa samples from individuals treated with antibiotic, probiotic and control diets to determine differentially expressed (DE) host genes. Fifty DE host genes were selected for further characterization using nanofluidic qPCR chips. We used 16S rRNA gene metabarcoding to characterize the rearing water and host gut microbiome (bacterial) communities. Daily administration of antibiotics and probiotics resulted in significant changes in fish gut and aquatic microbiota as well as more than 100 DE genes in the antibiotic and probiotic treatment fish, relative to healthy controls. Normal microbiota depletion by antibiotics mostly led to downregulation of different aspects of immunity and upregulation of apoptotic process. In the probiotic treatment, genes related to post-translation modification and inflammatory responses were up-regulated relative to controls. Our qPCR results revealed significant effects of treatment (antibiotic and probiotic) on rabep2, aifm3, manf, prmt3 gene transcription. Moreover, we found significant associations between members of Lactobacillaceae and Bifidobacteriaceae with host gene expression patterns. Overall, our analysis showed that the microbiota had significant impacts on many host signalling pathways, specifically targeting immune, developmental and metabolic processes. Our characterization of some of the molecular mechanisms involved in microbiome-host interactions will help develop new strategies for preventing/ treating microbiome disruption-related diseases.

Divergent RNA viruses infecting sea lice, major ectoparasites of fish.

Sea lice, the major ectoparasites of fish, have significant economic impacts on wild and farmed finfish, and have been implicated in the decline of wild salmon populations. As blood-feeding arthropods, sea lice may also be reservoirs for viruses infecting fish. However, except for two groups of negative-strand RNA viruses within the order Mononegavirales, nothing is known about viruses of sea lice. Here, we used transcriptomic data from three key species of sea lice (Lepeophtheirus salmonis, Caligus clemensi, and Caligus rogercresseyi) to identify 32 previously unknown RNA viruses. The viruses encompassed all the existing phyla of RNA viruses, with many placed in deeply branching lineages that likely represent new families and genera. Importantly, the presence of canonical virus-derived small interfering RNAs (viRNAs) indicates that most of these viruses infect sea lice, even though in some cases their closest classified relatives are only known to infect plants or fungi. We also identified both viRNAs and PIWI-interacting RNAs (piRNAs) from sequences of a bunya-like and two qin-like viruses in C. rogercresseyi. Our analyses showed that most of the viruses found in C. rogercresseyi occurred in multiple life stages, spanning from planktonic to parasitic stages. Phylogenetic analysis revealed that many of the viruses infecting sea lice were closely related to those that infect a wide array of eukaryotes with which arthropods associate, including fungi and parasitic tapeworms, implying that over evolutionary time there has been cross-phylum and cross-kingdom switching of viruses between arthropods and other eukaryotes. Overall, this study greatly expands our view of virus diversity in crustaceans, identifies viruses that infect and replicate in sea lice, and provides evidence that over evolutionary time, viruses have switched between arthropods and eukaryotic hosts in other phyla and kingdoms.

The effects of host quantitative genetic architecture on the gut microbiota composition of Chinook salmon (Oncorhynchus tshawytscha).

The microbiota consists of microbes living in or on an organism and has been implicated in host health and function. Environmental and host-related factors were shown to shape host microbiota composition and diversity in many fish species, but the role of host quantitative architecture across populations and among families within a population is not fully characterized. Here, Chinook salmon were used to determine if inter-population differences and additive genetic variation within populations influenced the gut microbiota diversity and composition. Specifically, hybrid stocks of Chinook salmon were created by crossing males from eight populations with eggs from an inbred line created from self-fertilized hermaphrodite salmon. Based on high-throughput sequencing of the 16S rRNA gene, significant gut microbial community diversity and composition differences were found among the hybrid stocks. Furthermore, additive genetic variance components varied among hybrid stocks, indicative of population-specific heritability patterns, suggesting the potential to select for specific gut microbiota composition for aquaculture purposes. Determining the role of host genetics in shaping their gut microbiota has important implications for predicting population responses to environmental changes and will thus impact conservation efforts for declining populations of Chinook salmon.

Molecular evidence for stress, inflammation and structural changes in non-specific ulcers in skin of farmed Chinook salmon (Oncorhynchus tshawytscha).

Fish skin is critical to physical defence against pathogens and there is a need to understand the physiological processes impacting ulcers and their healing. Ulcers have been reported in farmed Chinook salmon in New Zealand. This study investigated stress, immune and structural gene expression in farmed Chinook salmon skin with and without ulcers from two sites in New Zealand sampled from February (higher temperature, late summer) to May (lower temperature, late autumn). Skin samples taken adjacent to non-specific ulcers in May and control fish in February demonstrated upregulation of heat shock protein 70 relative to control fish in May. Anterior gradient 2 expression was upregulated in fish with ulcers relative to control fish (both February and May), suggesting increased mucous cell activity. Based on the results of this study, fish with non-specific ulcers showed evidence of stress, inflammation, re-epithelisation, and delayed healing near the ulcer site, elucidating the importance of these processes in the pathogenesis of non-specific ulcers in farmed chinook salmon.

Functional validation of transposable element-derived cis-regulatory elements in Atlantic salmon.

Transposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole-genome duplications (WGDs), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ∼100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. Here, we evaluated the gene-regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-CREs upstream of transcriptional start sites increased expression significantly. Our results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

Genomic variation across Chinook salmon populations reveals effects of a duplication on migration alleles and supports fine scale structure.

The distribution of ecotypic variation in natural populations is influenced by neutral and adaptive evolutionary forces that are challenging to disentangle. This study provides a high-resolution portrait of genomic variation in Chinook salmon (Oncorhynchus tshawytscha) with emphasis on a region of major effect for ecotypic variation in migration timing. With a filtered data set of ~13 million single nucleotide polymorphisms (SNPs) from low-coverage whole genome resequencing of 53 populations (3566 barcoded individuals), we contrasted patterns of genomic structure within and among major lineages and examined the extent of a selective sweep at a major effect region underlying migration timing (GREB1L/ROCK1). Neutral variation provided support for fine-scale structure of populations, while allele frequency variation in GREB1L/ROCK1 was highly correlated with mean return timing for early and late migrating populations within each of the lineages (r2  = .58-.95; p < .001). However, the extent of selection within the genomic region controlling migration timing was much narrower in one lineage (interior stream-type) compared to the other two major lineages, which corresponded to the breadth of phenotypic variation in migration timing observed among lineages. Evidence of a duplicated block within GREB1L/ROCK1 may be responsible for reduced recombination in this portion of the genome and contributes to phenotypic variation within and across lineages. Lastly, SNP positions across GREB1L/ROCK1 were assessed for their utility in discriminating migration timing among lineages, and we recommend multiple markers nearest the duplication to provide highest accuracy in conservation applications such as those that aim to protect early migrating Chinook salmon. These results highlight the need to investigate variation throughout the genome and the effects of structural variants on ecologically relevant phenotypic variation in natural species.

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines.

Finfish production has seen over three-fold increase in the past 30 years (1990-2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci-cr2 and mmp9 of A. salmon-were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.

The genomic basis of reproductive and migratory behaviour in a polymorphic salmonid.

Recent ecotypic differentiation provides unique opportunities to investigate the genomic basis and architecture of local adaptation, while offering insights into how species form and persist. Sockeye salmon (Oncorhynchus nerka) exhibit migratory and resident ("kokanee") ecotypes, which are further distinguished into shore-spawning and stream-spawning reproductive ecotypes. Here, we analysed 36 sockeye (stream-spawning) and kokanee (stream- and shore-spawning) genomes from a system where they co-occur and have recent common ancestry (Okanagan Lake/River in British Columbia, Canada) to investigate the genomic basis of reproductive and migratory behaviour. Examination of the genomic landscape of differentiation, differences in allele frequencies and genotype-phenotype associations revealed three main blocks of sequence differentiation on chromosomes 7, 12 and 20, associated with migratory behaviour, spawning location and spawning timing. Structural variants identified in these same areas suggest they could contribute to ecotypic differentiation directly as causal variants or via maintenance of their genomic architecture through recombination suppression mechanisms. Genes in these regions were related to spatial memory and swimming endurance (SYNGAP, TPM3), as well as eye and brain development (including SIX6), potentially associated with differences in migratory behaviour and visual habitats across spawning locations, respectively. Additional genes (GREB1L, ROCK1) identified here have been associated with timing of migration in other salmonids and could explain variation in timing of O. nerka spawning. Together, these results based on the joint analysis of sequence and structural variation represent a significant advance in our understanding of the genomic landscape of ecotypic differentiation at different stages in the speciation continuum.

Chinook salmon diversity contributes to fishery stability and trade-offs with mixed-stock harvest.

Variation among populations in life history and intrinsic population characteristics (i.e., population diversity) helps maintain resilience to environmental change and dampen interannual variability in ecosystem services. As a result, ecological variation, and the processes that generate it, is considered central to strategies for managing risks to ecosystems in an increasingly variable and uncertain world. However, characterizing population diversity is difficult, particularly in large and remote regions, which often prevents its formal consideration in management advice. We combined genetic stock identification of archived scale and tissue samples with state-space run-reconstruction models to estimate migration timing and annual return abundance for eight geographically and genetically distinct Chinook salmon populations within the Canadian portion of the Yukon River. We found that among-population variation in migration timing and return abundances resulted in aggregate return migrations that were 2.1 times longer and 1.4 times more stable than if they had composed a single homogeneous population. We then fit state-space spawner-recruitment models to the annual return abundances to characterize among-population diversity in intrinsic productivity and population size and their consequences for the fisheries they support. Productivity and carrying capacity varied among populations by approximately 2.4-fold (2.9 to 6.9 recruits per spawner) and three-fold (8800 to 27,000 spawners), respectively. This diversity implies an equilibrium trade-off between harvesting of the population aggregate and the conservation of individual populations whereby the harvest rate predicted to maximize aggregate harvests comes at the cost of overfishing ~40% of the populations but with a relatively low risk of extirpating the weakest ones. Our findings illustrate how population diversity in one of the largest salmon-producing river basins in the world contributes to fishery stability and food security in a region where salmon have high cultural and subsistence value. More generally, our work demonstrates the utility of molecular analyses of archived biological material for characterizing diversity in biological systems and its benefits and consequences for trade-offs in decision-making.

Broad-scale eDNA sampling for describing aquatic species distributions in running waters: Pacific lamprey Entosphenus tridentatus in the upper Snake River, USA.

One of the most fundamental yet challenging tasks for aquatic ecologists is to precisely delineate the range of species, particularly those that are broadly distributed, require specialized sampling methods, and may be simultaneously declining and increasing in different portions of their range. An exemplar is the Pacific lamprey Entosphenus tridentatus, a jawless anadromous fish of conservation concern that is actively managed in many coastal basins in western North America. To efficiently determine its distribution across the accessible 56,168 km of the upper Snake River basin in the north-western United States, we first delimited potential habitat by using predictions from a species distribution model based on conventionally collected historical data and from the distribution of a potential surrogate, Chinook salmon Oncorhynchus tshawytscha, which yielded a potential habitat network of 10,615 km. Within this area, we conducted a two-stage environmental DNA survey involving 394 new samples and 187 archived samples collected by professional biologists and citizen scientists using a single, standardized method from 2015 to 2021. We estimated that Pacific lamprey occupied 1875 km of lotic habitat in this basin, of which 1444 km may have been influenced by recent translocation efforts. Pacific lamprey DNA was consistently present throughout most river main stems, although detections became weaker or less frequent in the largest and warmest downstream channels and near their headwater extent. Pacific lamprey were detected in nearly all stocked tributaries, but there was no evidence of indigenous populations in such habitats. There was evidence of post-stocking movement because detections were 1.8-36.0 km upstream from release sites. By crafting a model-driven spatial sampling template and executing an eDNA-based sampling campaign led by professionals and volunteers, supplemented by previously collected samples, we established a benchmark for understanding the current range of Pacific lamprey across a large portion of its range in the interior Columbia River basin. This approach could be tailored to refine range estimates for other wide-ranging aquatic species of conservation concern.

Transcriptome profiling of the early developmental stages in the giant mussel Choromytilus chorus exposed to delousing drugs.

The giant mussel Choromytilus chorus is a marine bivalve commonly collected in central - southern Chile from fishery zones shared with the salmon industry. These economically relevant areas are also affected by the use of pesticides for controlling sea lice infestations in salmon aquaculture. Their main target is the sea louse Caligus rogercresseyi. However, other than some physiological impacts, the molecular effects of delousing drugs in non-target species such as C. chorus remain largely understudied. This study aimed to explore the transcriptome modulation of Trochophore and D larvae stages of C. chorus after exposure to azamethiphos and deltamethrin drugs. Herein, RNA-seq analyses and mRNA-lncRNAs molecular interactions were obtained. The most significant changes were found between different larval development stages exposed to delousing drugs. Notably, significant transcriptional variations were correlated with the drug concentrations tested. The biological processes involved in the development, such as cell movement and transcriptional activity, were mainly affected. Long non-coding RNAs (lncRNAs) were also identified in this species, and the transcription activity showed similar patterns with coding mRNAs. Most of the significantly expressed lncRNAs were associated with genes annotated to matrix metalloproteinases, collagenases, and transcription factors. This study suggests that exposure to azamethiphos or deltamethrin drugs can modulate the transcriptome signatures related to the early development of the giant mussel C. chorus.

Key role of mitochondrial mutation Leu107Ser (COX1) in deltamethrin resistance in salmon lice (Lepeophtheirus salmonis).

The pyrethroid deltamethrin (DTM) is used to treat Atlantic salmon (Salmo salar) against salmon louse (Lepeophtheirus salmonis) infestations. However, DTM resistance has evolved in L. salmonis and is currently common in the North Atlantic. This study aimed to re-assess the association between DTM resistance and mitochondrial (mtDNA) mutations demonstrated in previous reports. Among 218 L. salmonis collected in Scotland in 2018-2019, 89.4% showed DTM resistance in bioassays, while 93.6% expressed at least one of four mtDNA single nucleotide polymorphisms (SNPs) previously shown to be resistance associated. Genotyping at further 14 SNP loci allowed to define three resistance-associated mtDNA haplotypes, named 2, 3 and 4, occurring in 72.0%, 14.2% and 7.3% of samples, respectively. L. salmonis strains IoA-02 (haplotype 2) and IoA-10 (haplotype 3) both showed high levels (~ 100-fold) of DTM resistance, which was inherited maternally in crossing experiments. MtDNA haplotypes 2 and 3 differed in genotype for 17 of 18 studied SNPs, but shared one mutation that causes an amino acid change (Leu107Ser) in the cytochrome c oxidase subunit 1 (COX1) and was present in all DTM resistant while lacking in all susceptible parasites. We conclude that Leu107Ser (COX1) is a main genetic determinant of DTM resistance in L. salmonis.