There is growing recognition of the need to understand the mechanisms underlying organismal resilience (i.e. tolerance, acclimatization) to environmental change to support the conservation management of sensitive and economically important species. Here, we discuss how functional genomics can be used in conservation biology to provide a cellular-level understanding of organismal responses to environmental conditions. In particular, the integration of transcriptomics with physiological and ecological research is increasingly playing an important role in identifying functional physiological thresholds predictive of compensatory responses and detrimental outcomes, transforming the way we can study issues in conservation biology. Notably, with technological advances in RNA sequencing, transcriptome-wide approaches can now be applied to species where no prior genomic sequence information is available to develop species-specific tools and investigate sublethal impacts that can contribute to population declines over generations and undermine prospects for long-term conservation success. Here, we examine the use of transcriptomics as a means of determining organismal responses to environmental stressors and use key study examples of conservation concern in fishes to highlight the added value of transcriptome-wide data to the identification of functional response pathways. Finally, we discuss the gaps between the core science and policy frameworks and how thresholds identified through transcriptomic evaluations provide evidence that can be more readily used by resource managers.

Suboptimal fish stocking densities in experimental systems may elicit stress responses that can affect experimental results. Fish species, age and size, water chemistry and flow, and physical characteristics of the experimental system (e.g., tank, cage) are among the parameters to be considered when determining stocking densities. However, systematic studies to define fish densities minimizing stress in experimental systems are rarely performed. This is particularly true when working with species of low aquaculture value or a non-model test species such as the Delta Smelt (. Hypomesus transpacificus). The aim of this study was to use physiological stress biomarkers to determine suitable fish densities for specific experimental vessels routinely used for this species. We maintained late larval Delta Smelt (60. days post-hatch; dph) over a period of 24. h, at five different densities: 7, 14, 28, 42, and 56 fish per 8. L circular fish tank. We assessed whole body cortisol and transcriptomic biomarkers that lead to cortisol production to quantify stress levels. Both marker types delivered similar results. Cortisol levels were lowest at densities of 28 and 42 fish per tank, whereas lowest fish densities (7 and 14 fish per tank) evoked the highest stress levels. Genes such as Mineralocorticoid Receptor 1 and Glucocorticoid Receptor 2, as well as 11-Beta-Hydroxysteroid-Dehydrogenase-2 depicted the lowest expression levels at stocking densities 28 and 42, and elevated expression levels for stocking densities 7 and 14. Our data support the observations that late larval Delta Smelt should be exposed, acclimated, and cultured in groups rather than as individuals or in low numbers. This study indicates the importance of adequately defining experimental conditions that minimize stress, specifically when stress is measured as an endpoint. In addition to classical cortisol measurements, responses of the transcriptome also appear suitable in assessing stress responses in fish, and in determining optimal holding conditions, particularly if short-term responses are the study focus.Statement of relevance. The study highlights the importance of evaluating stress in order to determine species-specific stocking densities. The results are thus relevant to a wide audience in the field of aquaculture and experimental biology.

Turbidity can influence trophic levels by altering species composition and can potentially affect fish feeding strategies and predator-prey interactions. The estuarine turbidity maximum, described as an area of increased suspended particles, phytoplankton and zooplankton, generally represents a zone with higher turbidity and enhanced food sources important for successful feeding and growth in many fish species. The delta smelt (Hypomesus transpacificus) is an endangered, pelagic fish species endemic to the San Francisco Estuary and Sacramento-San Joaquin River Delta, USA, where it is associated with turbid waters. Turbidity is known to play an important role for the completion of the species' life cycle; however, turbidity ranges in the Delta are broad, and specific requirements for this fish species are still unknown. To evaluate turbidity requirements for early life stages, late-larval delta smelt were maintained at environmentally relevant turbidity levels ranging from 5 to 250 nephelometric turbidity units (NTU) for 24 h, after which a combination of physiological endpoints (molecular biomarkers and cortisol), behavioural indices (feeding) and whole-organism measures (survival) were determined. All endpoints delivered consistent results and identified turbidities between 25 and 80 NTU as preferential. Delta smelt survival rates were highest between 12 and 80 NTU and feeding rates were highest between 25 and 80 NTU. Cortisol levels indicated minimal stress between 35 and 80 NTU and were elevated at low turbidities (5, 12 and 25 NTU). Expression of stress-related genes indicated significant responses for gst, hsp70 and glut2 in high turbidities (250 NTU), and principal component analysis on all measured genes revealed a clustering of 25, 35, 50 and 80 NTU separating the medium-turbidity treatments from low- and high-turbidity treatments. Taken together, these data demonstrate that turbidity levels that are either too low or too high affect delta smelt physiological performance, causing significant effects on overall stress, food intake and mortality. They also highlight the need for turbidity to be considered in habitat and water management decisions.

The National Science Foundation and other funding agencies are increasingly requiring broader impacts in grant applications to encourage US scientists to contribute to science education and society. Concurrently, national science education standards are using more inquiry-based learning (IBL) to increase students' capacity for abstract, conceptual thinking applicable to real-world problems. Scientists are particularly well suited to engage in broader impacts via science inquiry outreach, because scientific research is inherently an inquiry-based process. We provide a practical guide to help scientists overcome obstacles that inhibit their engagement in K-12 IBL outreach and to attain the accrued benefits. Strategies to overcome these challenges include scaling outreach projects to the time available, building collaborations in which scientists' research overlaps with curriculum, employing backward planning to target specific learning objectives, encouraging scientists to share their passion, as well as their expertise with students, and transforming institutional incentives to support scientists engaging in educational outreach.

Obtaining robust estimates of population abundance is a central challenge hindering the conservation and management of many threatened and exploited species. Close-kin mark-recapture (CKMR) is a genetics-based approach that has strong potential to improve the monitoring of data-limited species by enabling estimates of abundance, survival, and other parameters for populations that are challenging to assess. However, CKMR models have received limited sensitivity testing under realistic population dynamics and sampling scenarios, impeding the application of the method in population monitoring programs and stock assessments. Here, we use individual-based simulation to examine how unmodeled population dynamics and aging uncertainty affect the accuracy and precision of CKMR parameter estimates under different sampling strategies. We then present adapted models that correct the biases that arise from model misspecification. Our results demonstrate that a simple base-case CKMR model produces robust estimates of population abundance with stable populations that breed annually; however, if a population trend or non-annual breeding dynamics are present, or if year-specific estimates of abundance are desired, a more complex CKMR model must be constructed. In addition, we show that CKMR can generate reliable abundance estimates for adults from a variety of sampling strategies, including juvenile-focused sampling where adults are never directly observed (and aging error is minimal). Finally, we apply a CKMR model that has been adapted for population growth and intermittent breeding to two decades of genetic data from juvenile lemon sharks (Negaprion brevirostris) in Bimini, Bahamas, to demonstrate how application of CKMR to samples drawn solely from juveniles can contribute to monitoring efforts for highly mobile populations. Overall, this study expands our understanding of the biological factors and sampling decisions that cause bias in CKMR models, identifies key areas for future inquiry, and provides recommendations that can aid biologists in planning and implementing an effective CKMR study, particularly for long-lived data-limited species.

As global change alters multiple environmental conditions, predicting species' responses can be challenging without understanding how each environmental factor influences organismal performance. Approaches quantifying mechanistic relationships can greatly complement correlative field data, strengthening our abilities to forecast global change impacts. Substantial salinity increases are projected in the San Francisco Estuary, California, due to anthropogenic water diversion and climatic changes, where the critically endangered delta smelt (Hypomesus transpacificus) largely occurs in a low-salinity zone (LSZ), despite their ability to tolerate a much broader salinity range. In this study, we combined molecular and organismal measures to quantify the physiological mechanisms and sublethal responses involved in coping with salinity changes. Delta smelt utilize a suite of conserved molecular mechanisms to rapidly adjust their osmoregulatory physiology in response to salinity changes in estuarine environments. However, these responses can be energetically expensive, and delta smelt body condition was reduced at high salinities. Thus, acclimating to salinities outside the LSZ could impose energetic costs that constrain delta smelt's ability to exploit these habitats. By integrating data across biological levels, we provide key insight into the mechanistic relationships contributing to phenotypic plasticity and distribution limitations and advance the understanding of the molecular osmoregulatory responses in nonmodel estuarine fishes.

Climate change is driving rapid changes in environmental conditions and affecting population and species' persistence across spatial and temporal scales. Integrating climate change assessments into biological resource management, such as conserving endangered species, is a substantial challenge, partly due to a mismatch between global climate forecasts and local or regional conservation planning. Here, we demonstrate how outputs of global climate change models can be downscaled to the watershed scale, and then coupled with ecophysiological metrics to assess climate change effects on organisms of conservation concern. We employed models to estimate future water temperatures (2010-2099) under several climate change scenarios within the large heterogeneous San Francisco Estuary. We then assessed the warming effects on the endangered, endemic Delta Smelt, Hypomesus transpacificus, by integrating localized projected water temperatures with thermal sensitivity metrics (tolerance, spawning and maturation windows, and sublethal stress thresholds) across life stages. Lethal temperatures occurred under several scenarios, but sublethal effects resulting from chronic stressful temperatures were more common across the estuary (median >60 days above threshold for >50% locations by the end of the century). Behavioral avoidance of such stressful temperatures would make a large portion of the potential range of Delta Smelt unavailable during the summer and fall. Since Delta Smelt are not likely to migrate to other estuaries, these changes are likely to result in substantial habitat compression. Additionally, the Delta Smelt maturation window was shortened by 18-85 days, revealing cumulative effects of stressful summer and fall temperatures with early initiation of spring spawning that may negatively impact fitness. Our findings highlight the value of integrating sublethal thresholds, life history, and in situ thermal heterogeneity into global change impact assessments. As downscaled climate models are becoming widely available, we conclude that similar assessments at management-relevant scales will improve the scientific basis for resource management decisions.

Climate change and associated increases in water temperatures may impact physiological performance in ectotherms and exacerbate endangered species declines. We used an integrative approach to assess the impact of elevated water temperature on two fishes of immediate conservation concern in a large estuary system, the threatened longfin smelt (Spirinchus thaleichthys) and endangered delta smelt (Hypomesus transpacificus). Abundances have reached record lows in California, USA, and these populations are at imminent risk of extirpation. California is currently impacted by a severe drought, resulting in high water temperatures, conditions that will become more common as a result of climate change. We exposed fish to environmentally relevant temperatures (14°C and 20°C) and used RNA sequencing to examine the transcriptome-wide responses to elevated water temperature in both species. Consistent with having a lower temperature tolerance, longfin smelt exhibited a pronounced cellular stress response, with an upregulation of heat shock proteins, after exposure to 20°C that was not observed in delta smelt. We detected an increase in metabolic rate in delta smelt at 20°C and increased expression of genes involved in metabolic processes and protein synthesis, patterns not observed in longfin smelt. Through examination of responses across multiple levels of biological organization, and by linking these responses to habitat distributions in the wild, we demonstrate that longfin smelt may be more susceptible than delta smelt to increases in temperatures, and they have little room to tolerate future warming in California. Understanding the species-specific physiological responses of sensitive species to environmental stressors is crucial for conservation efforts and managing aquatic systems globally.

California's coastal ecosystems are forecasted to undergo shifting ocean conditions due to climate change, some of which may negatively impact recreational and commercial fish populations. To understand if fish populations have the capacity to respond to multiple stressors, it is critical to examine interactive effects across multiple biological scales, from cellular metabolism to species interactions. This study examined the effects of CO₂-acidification and hypoxia on two naturally co-occurring species, juvenile rockfish (genus Sebastes) and a known predator, cabezon (Scorpaenichthys marmoratus). Fishes were exposed to two PCO₂ levels at two dissolved oxygen (DO) levels: ~600 (ambient) and ~1600 (high) μatm PCO₂ and 8.0 (normoxic) and 4.5 mg l^-1 DO (hypoxic) and assessments of cellular metabolism, prey behavior and predation mortality rates were quantified after 1 and 3 weeks. Physiologically, rockfish showed acute alterations in cellular metabolic enzyme activity after 1 week of acclimation to elevated PCO₂ and hypoxia that were not evident in cabezon. Alterations in rockfish energy metabolism were driven by increases in anaerobic LDH activity, and adjustments in enzyme activity ratios of cytochrome c oxidase and citrate synthase and LDH:CS. Correlated changes in rockfish behavior were also apparent after 1 week of acclimation to elevated PCO₂ and hypoxia. Exploration behavior increased in rockfish exposed to elevated PCO₂ and spatial analysis of activity indicated short-term interference with anti-predator responses. Predation rate after 1 week increased with elevated PCO₂; however, no mortality was observed under the multiple-stressor treatment suggesting negative effects on cabezon predators. Most noteworthy, metabolic and behavioral changes were moderately compensated after 3 weeks of acclimation, and predation mortality rates also decreased suggesting that these rockfish may be resilient to changes in environmental stressors predicted by climate models. Linking physiological and behavioral responses to multiple stressors is vital to understand impacts on populations and community dynamics.

Background

Transcriptomic data has demonstrated utility to advance the study of physiological diversity and organisms' responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations.

Results

We generated high quality blood transcriptome assemblies for hawksbill (Eretmochelys imbricata), loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance.

Conclusions

Obtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles.