The development and implementation of environmental DNA (eDNA) assays have revolutionized aquatic species monitoring, yet progress is impeded by inconsistent standards in assay design, validation, and reporting. When insufficiently validated quantitative PCR (qPCR) assays are used for management and conservation, false positives or false negatives can have significant ecological and policy consequences. Drawing on existing standardized workflows, we present an integrated, multi-stage decision-oriented framework for eDNA assay development and validation that spans conceptual design through to in situ testing. The first stage includes a decision checklist for in silico assay validation, adapted from the empirical Thalinger validation scale, which enables users to critically assess the risk of false positives and false negatives before entering the laboratory. This early-stage evaluation informs and strengthens subsequent in vitro (i.e., specificity, sensitivity, and interference) and in situ testing, the key principles of which are presented here in a streamlined and accessible manner to support both expert and non-expert users. We illustrate the flexibility and scalability of these guidelines through three diverse case studies, from regional validation of a published assay to design and validation of novel genus-specific and species-specific assays for application to water samples and dietary analysis, respectively. Our trainee-led initiative reflects interdisciplinary partnerships across academic institutions, government agencies, and conservation organizations, and demonstrates how co-produced molecular tools can generate actionable results for fisheries management. The resulting decision support tool offers a reproducible, transparent, and adaptable pipeline for assay development, with broad applicability across freshwater fish monitoring programs in Canada and beyond.

Carmine shiner Notropis percobromus is a small minnow found throughout the eastern USA, but its known distribution in Canada is limited to southeastern Manitoba. Listed as Endangered on Canada’s Species at Risk Act (SARA), it has been the focus of targeted sampling by Fisheries and Oceans Canada, and continued monitoring is important to test for (1) potential range contractions due to habitat loss and pollution and (2) a northward shift in distribution with climate change. To complement traditional capture-based sampling, we developed and validated a species-specific TaqMan probe-based quantitative PCR environmental DNA (eDNA) assay targeting a 137-base pair fragment of the mitochondrial cytochrome b gene. Genetic sequences from 50 non-target cypriniform species were used in silico to design the putative species-specific assay, and in vitro testing using tissue-derived DNA from 15 other leuciscid species in Manitoba confirmed amplification only with carmine shiner DNA. Field testing within and adjacent to the known range of carmine shiner in the Winnipeg River system yielded results consistent with collection records of the species, with eDNA detected in the Whitemouth, Birch, and Boggy rivers and no detections outside the known range. This assay therefore offers a non-invasive method for inferring the presence of this endangered fish; it can be used in a complementary manner with capture-based sampling to inform on changes in distribution (e.g., to non-invasively survey in peripheral areas of the species’ range and at sites of greater concern for anthropogenic disturbance), with confirmatory capture-based sampling focusing on relative abundance and population trends.

Highlights• Artificial fertilization produced sea lamprey larvae across three brood years.• Fish meal-supplemented yeast improved first-year growth and survival.• Very high larval density caused complete mortality by 90 days.• Larval growth declined strongly as rearing density increased.• Laboratory rearing produced faster age-1 growth than field estimates.

The second iteration of the international conference "Pathway to Increase Standards and Competency of eDNA Surveys" was held at the University of Guelph, Guelph, Ontario, Canada from 18 June to 20 June 2023. During this environmental DNA (eDNA) conference, 60 oral and 25 poster presentations from academia, government, industry, NGOs, and Indigenous partners discussed the latest developments in eDNA research, explored strategies to inform public policy, and presented future directions in the field. The conference also included three panel discussions focused on prominent themes in the eDNA space, and five workshops dedicated to practical eDNA tools and methods. Recordings of presentations at the conference have been made available on YouTube. Here we summarize the major themes covered during the conference, provide our concluding remarks, and share the conference abstracts.

The benefits to humans of living by the ocean have led many coastal settlements to grow into large, densely populated cities. Large coastal cities have had considerable environmental effects on marine ecosystems through resource extraction, waste disposal, coastal development, and trade and travel routes. While our understanding of the ecological and evolutionary consequences of urbanisation for wildlife in terrestrial systems has received considerable recent attention, the consequences of urbanisation in marine systems are not well known. Using microsatellite datasets associated with published research on marine fish population genetics, we built a global database of genotypic data spanning 75,361 individuals sampled from 73 species at 1085 sample sites throughout the world's oceans. We found that genetic diversity and effective population sizes were significantly lower at marine fish sample sites associated with denser human populations, regardless of species and locality. The loss of genetic diversity near denser human populations indicates habitats near human settlements are less able to support large populations. Small effective population sizes, in turn, dampen the efficiency of natural selection near dense urban settlements. The loss of genetic diversity near cities is concerning for maintaining functioning marine ecosystems and sustainable fisheries. Our work highlights the need to mitigate environmental threats from human activities and focus efforts on sustainable urban planning and resource use to conserve marine biodiversity and sustain coastal fisheries and ecosystems.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America is among the largest and most successful control programs of an invasive species anywhere on the planet. The effort began more than 75 years ago; it unites multiple nations, states, and provinces with the common goal of controlling this invasive species and protecting a valuable fishery. The science-based control program is administered by the Great Lakes Fishery Commission (GLFC), a body arising from a treaty signed by the United States and Canada. In the present article, we share 10 lessons learned from decades of successful sea lamprey control with the hopes of informing ongoing and future control programs targeting biological invasions. The 10 lessons we identified are to act boldly in times of crisis, to maintain the social license, to invest in capacity building, to break down the silos, to support fundamental science, to diversify your portfolio of control measures, to strive for continuous improvement, to confront the trade-off between information and action, to keep your foot on the gas, and to keep your eyes on the prize. The GLFC has long fostered a framework that uses some military strategy and verbiage that extends across the lessons (e.g., know your enemy). Other lessons are more nascent as the GLFC reenvisions its relationship with Indigenous peoples and governments in a path to reconciliation where two-eyed seeing is being embraced. Through adaptive management, horizon scanning methods, and embracing implementation science, the lessons learned about sea lamprey control will continue to evolve, which is itself a lesson. We submit that the lessons shared in the present article will help guide invasive species control programs spanning taxa, ecosystems, and regions.

Deepwater sculpin Myoxocephalus thompsonii is found in the Laurentian Great Lakes and deep, cold, oxygenated, oligotrophic lakes from the Gatineau region of Quebec to Great Slave and Great Bear lakes in northwestern Canada. Its distribution along the path of glacial retreat is patchy, although unknown populations may exist given the challenges of sampling deep lakes, especially in remote locations. Deepwater sculpin is of conservation concern, with two of four known populations in Quebec having been extirpated due to eutrophication, and the Great Lakes–Upper St. Lawrence population is listed as Special Concern under Canada’s Species at Risk Act. In this collaborative project between the Gatineau Fish and Game Club, SOI Foundation, Ottawa Riverkeeper, Canadian Museum of Nature, University of Manitoba, and GEN-FISH, we tested the ability of environmental DNA (eDNA) protocols to detect this species in 31 Mile Lake, Quebec, where it is known to occur. We then applied these methods to test for potential novel detections in Lac Pémichangan, which is adjacent but not connected to 31 Mile Lake. Deepwater sculpin eDNA was detected in water collected below the thermocline in August or September at 7/8 (2021), 5/5 (2022), and 2/6 (2023) sites in 31 Mile Lake. However, although detections in 2/162 quantitative PCR (qPCR) replicates were recorded in Lac Pémichangan in 2022, follow-up qPCRs suggested contamination. These eDNA protocols could be used to survey other candidate lakes for the presence of this elusive deepwater species and to monitor lakes where it is threatened by eutrophication, climate warming, or invasive species.

The Arctic is warming at an unprecedented rate and with longer growing seasons, greater rainfall, and less snowfall. Cold-adapted ectotherms, such as the Arctic charr, Salvelinus alpinus (Linnaeus 1758), are likely to experience changes to growth as a result. Anadromous Arctic charr (charr, hereafter) are of great importance for northern communities, providing a source of income from commercial fisheries and food security from subsistence harvest. Initially, warming is expected to increase the growth of charr, benefitting subsistence and commercial fisheries in the short term. However, over longer time scales, temperatures exceeding the optimum for growth will likely result in metabolic stress, slowed growth, and higher mortality. Thus, the long-term consequences of climate change will likely be negative. We assessed anadromous charr growth from 1984 to 2013 in three stocks around Cumberland Sound using otolith measurements as proxies for age-specific growth. Trend analyses indicated growth had increased in pre-migratory ages over the years. We used mixed models to investigate changes to growth for ages 1–10 in relation to climate variables, finding that growing degree days had the greatest positive influence on ages 1–6 while annual precipitation had an overall negative effect on growth in ages 1–2 and 6–10. Contrary to previous assessments on these stocks, our results suggest charr have indeed experienced changes to growth with climate change. These findings emphasize the need for more thorough long-term growth studies in the management of fisheries as altered growth will affect food security and the economy across the Canadian Arctic.

Background: Environmental DNA (eDNA) sampling and analysis have the potential to revolutionize species monitoring, but the effective implementation in field conditions remains uncertain. The current study addresses this knowledge gap by developing a robust eDNA sampling protocol for the detection of larval Sea Lamprey Petromyzon marinus in Great Lakes tributaries. Methods: Three experiments were conducted to optimize eDNA field sample collection. The first experiment compared the performance of 0.45-μm, 1.2-μm, 5.0-μm cellulose nitrate (CN) filters and a 1.5-μm glass-fiber filter to determine which filter consistently yielded the highest median DNA copy number. The second experiment evaluated the performance of two filtration devices for eDNA sample collection and filtration, an autosampler (Halltech OSMOS aquatic eDNA sampler, Halltech Environmental and Aquatic Research, Guelph, Ontario, Canada) and a handheld peristaltic pump. In the third experiment, a biweekly eDNA survey was conducted to investigate the temporal dynamics of spawning Sea Lamprey eDNA to determine at what point during the season only larval lamprey eDNA is detected. Results: Our findings indicate that CN filters with a pore size of 1.2 μm or 5.0 μm captured consistently the highest amount of eDNA, but the 5.0-μm CN filter was selected for routine use due to its superior performance and reduced risk of clogging. We found no significant performance differences between the OSMOS aquatic eDNA sampler and the peristaltic pump across three response variables (frequency of contaminated field negative controls, PCR inhibition, and positive detections), suggesting both devices can reliably be used. Moreover, our study found that the spawning Sea Lamprey eDNA signal attenuates approximately 4–6 weeks after the last adult Sea Lamprey capture, which is consistent with previous research. Discussion: By synthesizing the results, we provide a streamlined eDNA sampling protocol for larval Sea Lamprey monitoring. We recommend beginning eDNA sampling at least six weeks after the end of the estimated regional spawning period and using a 5.0-μm CN filter in combination with the OSMOS aquatic eDNA sampler, with the handheld peristaltic pump serving as a backup. This optimized approach improves the efficacy and reliability of eDNA-based monitoring.

Aim: Local species distributions are often geographically restricted to a subset of environmental conditions across a species' full range, complicating forecasting climate warming effects. However, Bayesian species distribution models (SDM) can leverage geographically restricted datasets with broader knowledge of habitat relationships across the species' range to forecast climate vulnerability in data-limited regions. Location: Northern Canada. Methods: Principles of niche tracking and niche expansion were explored using an innovative Bayesian SDM approach to refine a climate vulnerability assessment for bull trout (Salvelinus confluentus), a cold-water riverine fish. The SDM was fit to a large, spatially dense fish occurrence and stream temperature dataset to model how climatic and geomorphic factors influence the current and future distribution of bull trout near its northern range extent. To assess niche tracking, wherein modelled relationships were based on observed occurrence patterns, we fitted the SDM with uninformative priors. For niche expansion, which assumes the population can adjust to occupy a warmer niche like southerly populations, we added an informative prior for summer stream water temperature occupancy. Models projected effects of warming on the distribution of suitable habitat using Representative Concentration Pathways 4.5 and 8.5 emissions scenarios for 2061-2080. Results: Bull trout distribution was patchy and limited to intermediate thermal and slope conditions in streams with high groundwater contributions. The latter is a key determinant of biogeographic patterns not seen elsewhere across the species' range. Under niche tracking, suitable habitat extent is projected to decline by 36%-46%, while under niche expansion, suitable habitat could increase by 25%-28%. Main Conclusions: The large dichotomy between projections illustrates the importance of considering local features and adaptive capacity when forecasting potential responses of cold-water fishes to climate warming. It also highlights a need for studies to better understand the mechanisms that may prevail as species distributions shift this century.

All vertebrate adaptive immune systems exhibit distinct lymphocyte lineages. In jawless vertebrates, such as lampreys, T-like cells are thought to develop in lympho-epithelial structures at the tips of gill filaments, termed thymoids. However, it is unclear whether thymoids are functionally equivalent to the thymus of jawed vertebrates. Indeed, because the structural modules that are somatically assembled to form the antigen receptors of jawless and jawed vertebrates differ, development and selection of T cells may be governed by clade-specific genetic networks. To address this question, we have replaced the mouse Foxn1 gene, a key regulator of the thymic microenvironment in jawed vertebrates, with the orthologous FOXN1 lamprey gene, which is expressed in the thymoids alongside genes orthologous to known targets of the mouse Foxn1 transcription factor. The reconstituted thymi support normal T cell development, and, to a lesser extent, also support B cell development, indicating that the lamprey FOXN1 gene can rescue the block of thymic epithelial cell differentiation in mice deficient for the endogenous Foxn1 gene. The absence of overt autoimmunity in transgenic mice suggests that the reconstituted thymic microenvironment directs the development of a self-tolerant T cell repertoire. These findings highlight the remarkable similarity of thymic epithelial functions in jawed and jawless vertebrates, despite more than 500 My of independent evolution. Our results thus suggest that the emergence of the Foxn1 transcription factor in the common ancestor of vertebrates was associated with the advent of a specialized tissue environment supporting the development and selection of T cells.

For decades, many freshwater fishes native to North America have been written off as “rough fish” and neglected as inconsequential or even scorned if perceived as inimical to traditional gamefish. Across the continent, the exact species included in this category vary but often include members of the families Lepisosteidae, Amiidae, Hiodontidae, Catostomidae, Ictaluridae, and Sciaenidae. The casual or callous treatment of these fishes, although occasionally questioned by diversity-minded scientists and the public for at least a century, has recently come under more widespread scientific scrutiny, as more holistic views of ecosystems and ecosystem-based management are increasingly embraced and implemented. Paradoxically, we are also in an era where increasing technological capabilities have exacerbated the casual disposal of native fishes via bowfishing and other methods, often followed by widespread publicity through social media. Amid this rapid technological evolution, recent scientific studies have identified and highlighted the long lifespans, erratic recruitment, and surprisingly complex stock composition and life histories of many of these neglected fishes. Conservation of these distinctive and fragile taxa has been initiated under the recognition of this life history diversity. As exemplified by the 23 articles in this Special Issue on “Underappreciated Native Fishes of North America and their Management” and highlighted in this introductory article, research on many of these taxa has surged, as has their need for management. Scientific discoveries during this timeframe have redefined our understanding of several of the species and have led to an enlightened appreciation of these fishes and their valuable roles in freshwater ecosystems.

Accurate taxonomy is fundamental to the study and conservation of biodiversity. Because of their morphological similarities, most brook and river lampreys in western North America have been placed in the genus Lampetra along with lampreys from Eurasia and eastern North America. However, molecular-based phylogenetic studies dating back several decades indicate that lampreys from Pacific drainages are genetically distinct from Atlantic Lampetra. Reviewing previous phylogenetic analysis of two mitochondrial and two nuclear genes for Northern Hemisphere lampreys, we assign these western North American brook and river lampreys to a new genus, Occidentis. To assess species diversity within Occidentis, we performed a species delimitation analysis using all publicly available cytochrome b sequences of the genus. Similar to previous studies, O. ayresii and O. richardsoni were not reciprocally monophyletic and are best categorized as life history variants of a single species. In addition to O. pacifica, O. hubbsi, and the diverse O. ayresii species complex, as many as seven undescribed candidate species from Oregon and California were identified, supporting results from previous studies with more geographically limited datasets. One specimen from Paynes Creek, California, was identified as a candidate species, although this single individual showed minimal interspecific divergence (1.34%) with O. hubbsi. Further genetic assessment along with information on morphology and phylogeography is needed to determine whether the variation observed between groups of candidate species represents distinct species or divergent lineages within a species complex. Additional sampling will inform whether there are additional species not currently represented in this dataset. Thus, the number of species formally recognized under Occidentis is subject to change with new information. Systematic assessment of the distribution and phylogenetic complexity within Occidentis will enhance our understanding of its evolutionary history and taxonomic diversity, which will guide efforts to conserve the biodiversity of lampreys.