Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.

Epidermal cell cultures from white and green sturgeon (Acipenser transmontanus and medirostris): Expression of TGM1-like transglutaminases and CYP4501A.

Using a system optimized for propagating human keratinocytes, culture of skin samples from white and green sturgeons generated epithelial cells capable of making cross-linked protein envelopes. Two distinct forms of TGM1-like mRNA were molecularly cloned from the cells of white sturgeon and detected in green sturgeon cells, accounting for their cellular envelope forming ability. The protein translated from each displayed a cluster of cysteine residues resembling the membrane anchorage region expressed in epidermal cells of teleosts and tetrapods. One of the two mRNA forms (called A) was present at considerably higher levels than the other (called B) in both species. Continuous lines of white sturgeon epidermal cells were established and characterized. Size measurements indicated that a substantial fraction of the cells became enlarged, appearing similar to squames in human epidermal keratinocyte cultures. The cultures also expressed CYP1A, a cytochrome P450 enzyme inducible by activation of aryl hydrocarbon receptor 2 in fish. The cells gradually improved in growth rate over a dozen passages while retaining envelope forming ability, TGM1 expression and CYP1A inducibility. These cell lines are thus potential models for studying evolution of fish epidermis leading to terrestrial adaptation and for testing sturgeon sensitivity to environmental stresses such as pollution.

Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time.

Environmental DNA (eDNA) analysis has gained traction as a precise and cost-effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA protocol. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank 27 different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an estuarine environment. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. We collected samples composed of 500 mL estuarine water from Deverton Slough (38°11'16.7"N 121°58'34.5"W) and 500 mL from tank water containing 1.3 juvenile Chinook Salmon per liter. Then, we compared extraction methods, filter types, use of inhibitor removal kit for DNA yield, processing time, and protocol cost. Lastly, we used an MCMC algorithm together with machine learning to understand the DNA yield of each step of the protocol as well as the interactions between those steps. Glass fiber filtration was to be the most resilient to high turbidites, filtering the samples in 2.32 ± 0.08 min instead of 14.16 ± 1.86 min and 6.72 ± 1.99 min for nitrocellulose and paper filter N1, respectively. The filtration DNA yield percentages for paper filter N1, glass fiber, and nitrocellulose were 0.00045 ± 0.00013, 0.00107 ± 0.00013, 0.00172 ± 0.00013. The DNA extraction yield percentage for QIagen, dipstick, NaOH, magnetic beads, and direct dipstick ranged from 0.047 ± 0.0388 to 0.475 ± 0.0357. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we found that a protocol combining glass filters, magnetic beads, and an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies in aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA protocol should be used under different study constraints.

Hepatobiliary Analyses Suggest Chronic PAH Exposure in Hakes (Urophycis spp.) Following the Deepwater Horizon Oil Spill.

Prior to the Deepwater Horizon oil spill, we lacked a comprehensive baseline of oil contamination in the Gulf of Mexico's sediments, water column, and biota. Gaps in prespill knowledge limit our ability to determine the aftereffects of the Deepwater Horizon blowout or prepare to mitigate similar impacts during future oil spill disasters. We examined spatiotemporal differences in exposure to and metabolism of polycyclic aromatic hydrocarbons (PAHs) in 2 hake species (Urophycis spp.) to establish a current baseline for these ecologically important, abundant, and at-risk demersal fishes. Gulf hake (Urophycis cirrata) and southern hake (Urophycis floridana) were collected throughout the Gulf of Mexico during extensive longline surveys from 2012 to 2015. Analyses of biliary PAH metabolites and liver PAH concentrations provided evidence of exposures to di- and tricyclic compounds, with the highest concentrations measured in the northern Gulf of Mexico. Species-specific differences were not detected, but temporal trends observed in biliary PAHs suggest a decrease in acute exposures, whereas increasing liver PAHs suggest chronic exposures marked by greater assimilation than metabolism rates. To our knowledge, the present study provides the first multitissue contaminant analyses, as well as the most exhaustive biometric analyses, for both gulf and southern hakes. Though sources of exposure are complex because of multiple natural and anthropogenic PAH inputs, these results will facilitate the development of much needed health metrics for Gulf of Mexico benthos. Environ Toxicol Chem 2019;38:2740-2749. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Triploidy in white sturgeon (Acipenser transmontanus): Effects of acute stress and warm acclimation on physiological performance.

Previous studies have demonstrated reduced performance in triploid fish when reared under suboptimal conditions, which may be the result of a higher susceptibility to stressors when compared to diploids. The goal of this project was to investigate differences in the capacity of diploid (8 N) and triploid (12 N) white sturgeon, Acipenser transmontanus, to respond to both warm acclimation (6-weeks of acclimation to either 18 or 22 °C) and a subsequent acute stress (10-min low water stress). Following the 6-week acclimation, fish were sampled either before or following an acute low water stress. Bioindices of the primary and secondary stress response, hematology and cellular metabolic status were measured. We also sought to determine if time to peak cortisol levels were similar between diploid and triploid sturgeon after exposure to a severe acute stressor (netting stress). While both ploidies had similar primary and secondary responses to acute stress, both with and without warm acclimation, warm acclimation impacted the ability of diploid and triploid white sturgeon to mount a typical stress response to an acute stressor. In response to warm acclimation, triploids exhibited little change in branchial lactate dehydrogenase activity, while diploids increased activity. After exposure to an acute water reduction stress, diploids increased citrate synthase activity, yet triploids showed a decrease in activity. Differences in metabolic enzyme activity in response to warm acclimation and acute stress suggest triploid white sturgeon may have a reduced cellular metabolic capacity under chronic and acute stress, which may impact performance of triploid sturgeon in suboptimal conditions.

The effects of warm temperature acclimation on constitutive stress, immunity, and metabolism in white sturgeon (Acipenser transmontanus) of different ploidies.

Previous studies suggest fish with additional copies of their genome (polyploids) underperform in suboptimal conditions and may be more susceptible to stress and disease. The objective of this study was to determine the role ploidy plays in the physiological response of white sturgeon to chronically elevated water temperatures. White sturgeon of two ploidies (8 N and 10 N) were acclimated to ambient (18 °C) and warm (22 °C) water. Bioindices of stress (plasma cortisol, glucose and lactate, total erythrocyte count, hematocrit, hemoglobin, mean erythrocyte volume, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration), immunity (respiratory burst, plasma lysozyme, and total leukocyte count), and cellular metabolic capacity (lactate dehydrogenase and citrate synthase activity) were measured before and after a 6-week acclimation period. Both ploidies appear comparable in their constitutive immune and stress parameters and respond similarly to warming. Hematological indices suggest 8 N and 10 N sturgeon are similar in oxygen carrying capacity; however, differences in enzyme activity between ploidies indicate that 10 N sturgeon may have a lower cellular aerobic capacity. Our results have implications for the screening and management of ploidy on white sturgeon farms and hatcheries, as the differences between ploidies may affect 10 N sturgeon performance at elevated water temperatures. Further research is needed to elucidate the differences in inducible stress and immune responses and metabolism of white sturgeon of different ploidies.

Research tools to investigate movements, migrations, and life history of sturgeons (Acipenseridae), with an emphasis on marine-oriented populations.

Worldwide, sturgeons (Acipenseridae) are among the most endangered fishes due to habitat degradation, overfishing, and inherent life history characteristics (long life span, late maturation, and infrequent spawning). As most sturgeons are anadromous, a considerable portion of their life history occurs in estuarine and marine environments where they may encounter unique threats (e.g., interception in non-target fisheries). Of the 16 marine-oriented species, 12 are designated as Critically Endangered by the IUCN, and these include species commercially harvested. We review important research tools and techniques (tagging, electronic tagging, genetics, microchemistry, observatory) and discuss the comparative utility of these techniques to investigate movements, migrations, and life-history characteristics of sturgeons. Examples are provided regarding what the applications have revealed regarding movement and migration and how this information can be used for conservation and management. Through studies that include Gulf (Acipenser oxyrinchus desotoi) and Green Sturgeon (A. medirostris), we illustrate what is known about well-studied species and then explore lesser-studied species. A more complete picture of migration is available for North American sturgeon species, while European and Asian species, which are among the most endangered sturgeons, are less understood. We put forth recommendations that encourage the support of stewardship initiatives to build awareness and provide key information for population assessment and monitoring.