Integrated organismal responses induced by projected levels of CO2 and temperature exposures in the early life stages of lake sturgeon.

Atmospheric CO2 and temperature are rising concurrently, and may have profound impacts on the transcriptional, physiological and behavioural responses of aquatic organisms. Further, spring snowmelt may cause transient increases of pCO2 in many freshwater systems. We examined the behavioural, physiological and transcriptomic responses of an ancient fish, the lake sturgeon (Acipenser fulvescens) to projected levels of warming and pCO2 during its most vulnerable period of life, the first year. Specifically, larval fish were raised in either low (16°C) or high (22°C) temperature, and/or low (1000 µatm) or high (2500 µatm) pCO2 in a crossed experimental design over approximately 8 months. Following overwintering, lake sturgeon were exposed to a transient increase in pCO2 of 10,000 µatm, simulating a spring melt based on data in freshwater systems. Transcriptional analyses revealed potential connections to otolith formation and reduced growth in fish exposed to high pCO2 and temperature in combination. Network analyses of differential gene expression revealed different biological processes among the different treatments on the edges of transcriptional networks. Na+/K+-ATPase activity increased in fish not exposed to elevated pCO2 during development, and mRNA abundance of the ß subunit was most strongly predictive of enzyme activity. Behavioural assays revealed a decrease in total activity following an acute CO2 exposure. These results demonstrate compensatory and compounding mechanisms of pCO2 and warming dependent on developmental conditions in lake sturgeon. Conserved elements of the cellular stress response across all organisms provide key information for how other freshwater organisms may respond to future climate change.

Elevated temperatures reduce population-specific transcriptional plasticity in developing lake sturgeon (Acipenser fulvescens).

Rising mean and variance in temperatures elevates threats to endangered freshwater species such as lake sturgeon, Acipenser fulvescens. Previous research demonstrated that higher temperatures during development result in physiological consequences for lake sturgeon populations throughout Manitoba, Canada, with alteration of metabolic rate, thermal tolerance, transcriptional responses, growth and mortality. We acclimated lake sturgeon (30-60 days post fertilization, a period of high mortality) from northern and southern populations (56°02'46.5″N, 96°54'18.6″W and 50°17'52″N, 95°32'51″W, respectively, separated by approximately 650 km) within Manitoba to current (summer highs of 20-23°C) and future projected (+2-3°C) environmental temperatures of 16, 20 and 24°C for 30 days, and we measured gill transcriptional responses using RNAseq. Transcripts revealed SNPs consistent with genetically distinct populations and transcriptional responses altered by acclimation temperature. There were a higher number of differentially expressed transcripts observed in the southern, compared to the northern, population as temperatures increased, indicating enhanced transcriptional plasticity. Both lake sturgeon populations responded to elevated acclimation temperatures by downregulating the transcription of genes involved in protein synthesis and energy production. Furthermore, there were population-specific thresholds for the downregulation of processes promoting transcriptional plasticity as well as mitochondrial function as the northern population showed decreases at 20°C, while this capacity was not diminished until 24°C in the southern population. These transcriptional responses highlight the molecular impacts of increasing temperatures for divergent lake sturgeon populations during vulnerable developmental periods and the critical influence of transcriptome plasticity on acclimation capacity.

Elevated temperatures dampen the innate immune capacity of developing lake sturgeon (Acipenser fulvescens).

Chronic exposure to high temperatures may leave freshwater fishes vulnerable to opportunistic pathogens, particularly during early life stages. Lake sturgeon, Acipenser fulvescens, populations within the northern expanse of their range in Manitoba, Canada, may be susceptible to high temperature stress and pathogenic infection. We acclimated developing lake sturgeon for 22 days to two ecologically relevant, summer temperatures (16 and 20°C). Individuals from both acclimation treatments were then exposed to 0, 30 and 60 µg ml-1 bacterial lipopolysaccharides (endotoxins), as an immune stimulus, for 48 h and sampled 4 and 48 h during trial exposures and following a 7 day recovery period. We then measured whole-body transcriptional (mRNA) responses involved in the innate immune, stress and fatty acid responses following acute exposure to the bacterial endotoxins. Data revealed that overall levels of mRNA transcript abundance were higher in 20°C-reared sturgeon under control conditions. However, following exposure to a bacterial stimulus, lake sturgeon acclimated to 16°C produced a more robust and persistent transcriptional response with higher mRNA transcript abundance across innate immune, stress and fatty acid responses than their 20°C-acclimated counterparts. Additional whole-animal performance metrics (critical thermal maximum, metabolic rate, cortisol concentration and whole-body and mucosal lysozyme activity) demonstrated acclimation-specific responses, indicating compromised metabolic, stress and enzymatic capacity following the initiation of immune-related responses. Our study showed that acclimation to 20°C during early development impaired the immune capacity of developing lake sturgeon as well as the activation of molecular pathways involved in the immune, stress and fatty acid responses. The present study highlights the effects of ecologically relevant, chronic thermal stress on seasonal pathogen susceptibility in this endangered species.

Gene expression and latitudinal variation in the stress response in Fundulus heteroclitus.

Atlantic killifish, Fundulus heteroclitus, are intertidal marsh fish found along the east coast of North America. Associated with the thermal gradient along this coast, northern and southern killifish populations are known to differ in morphology, behavior, and physiology, including in their cortisol stress response. Our goal was to explore population differences in the stress response and identify underlying molecular mechanisms. We measured responses to both acute and repeated stress in plasma cortisol, stress axis mRNA expression, and body condition in northern and southern killifish. Following an acute stressor, the southern population had higher cortisol levels than the northern population but there was no difference between populations following repeated stress. In the brain, both corticotropin releasing factor and its binding protein had higher expression in the southern than the northern population, but the northern population showed more changes in mRNA levels following a stressor. In the head kidney, Melanocortin 2 Receptor and steroidogenic acute regulatory protein mRNA levels were higher in the southern population suggesting a larger capacity for cortisol synthesis than in the northern fish. Lastly, the glucocorticoid receptor GR1 mRNA levels were greater in the liver of southern fish, suggesting a greater capacity to respond to cortisol, and GR2 had differential expression in the head kidney, suggesting an interpopulation difference in stress axis negative feedback loops. Southern, but not northern, fish were able to maintain body condition following stress, suggesting that these differences in the stress response may be important for adaptation across latitudes.

Long-term effects of temperature during early life on growth and fatty acid metabolism in age-0 Lake Sturgeon (Acipenser fulvescens).

Environmental temperature during early life may have prolonged effects on growth and fatty acid metabolism, which could strongly influence overwintering survival in the first year of life for temperate-zone fish. In the present study, we examined how temperature during early life history might influence growth performance and fatty acid metabolism in age-0 Lake Sturgeon (Acipenser fulvescens) when exposed to cold temperatures at later stages. Fish were initially at 16 °C and subsequently held at 16 °C or 20 °C for 60 days beginning at 34 days post fertilization (dpf). Then, all fish were subsequently raised at the same temperature of 16 °C until the onset of cold conditioning at 158 dpf where temperature was gradually decreased to 3.5 °C and remained there for two weeks. Samples were collected before (151 dpf) and after cold conditioning (199 dpf) to measure total length, body mass, whole body metabolic rate, fatty acid profile in phospholipids and triglycerides and mRNA expression of genes associated with fatty acid desaturation, elongation and ß-oxidation. Results revealed that before cold conditioning, total length and body mass did not differ between temperature groups, but fish raised at 20 °C showed a lower condition factor. During the cold conditioning, only fish raised at 16 °C grew significantly longer and heavier. There was no difference in metabolic rates between treatments. Significant increases in total monounsaturated fatty acids with decreases in total saturated fatty acids were identified in phospholipids and triglycerides in both temperature groups after the cold conditioning; however, the 20 °C group did not significantly increase levels of gene expression associated with fatty acid desaturation (SCD and FADS1) whereas the 16 °C group did. Our results suggest that thermal experience during early life may influence overwintering survival of age-0 Lake Sturgeon.

Survival and gene expression responses in immune challenged larval lake sturgeon.

Larval lake sturgeon, Acipenser fulvescens, reared in hatcheries for stock enhancement of wild populations may be susceptible to early opportunistic bacterial infection. Thus, we examined survival and whole-body mRNA expression of both stress- and immune-related genes (MyD88, IL-1ß, StAR, GR1, and HSP70) in 30 days post fertilization larval lake sturgeon following immune challenge with lipopolysaccharides (LPS). Larval sturgeon were exposed to 0, 25, 50, 100, 150, and 200 µg ml-1 LPS and sampled after 30 min, 4 h, and 48 h. Mortality was zero in 0 and 25 µg ml-1 LPS; 37.5% in 50 µg ml-1 LPS and 100% in the higher concentrations. Expression of MyD88 and StAR mRNA were positively correlated and increased with time in the 50 µg ml-1 LPS treatment. There was an influence of both treatment and time on IL-1ß mRNA, with expression 10-fold higher than controls after 4 h. Expression of HSP70 mRNA was suppressed within 30 min of 50 µg ml-1 LPS exposure and remained so throughout the time course. Correlated mRNA expression of GR1 with MyD88, StAR and IL-1ß suggests a potential relationship between the innate immune and glucocorticoid responses of larval lake sturgeon during this early developmental stage. Data presented suggest that larval lake sturgeon largely responded with predicted changes in gene expression of immune related and stress response genes following LPS challenge. This study provides a foundation for future research examining the effects of hatchery and naturally occurring stressors on the immune responses of larval lake sturgeon.

The effects of population and thermal acclimation on the growth, condition and cold responsive mRNA expression of age-0 lake sturgeon (Acipenser fulvescens).

In Manitoba, Canada, wild lake sturgeon (Acipenser fulvescens) populations exist along a latitudinal gradient and are reared in hatcheries to bolster threatened populations. We reared two populations of lake sturgeon, one from each of the northern and southern ends of Manitoba and examined the effects of typical hatchery temperatures (16°C) as well as 60-day acclimation to elevated rearing temperatures (20°C) on mortality, growth and condition throughout early development. Additionally, we examined the cold shock response, which may be induced during stocking, through the hepatic mRNA expression of genes involved in the response to cold stress and homeoviscous adaptation (HSP70, HSP90a, HSP90b, CIRP and SCD). Sturgeon were sampled after 1 day and 1 week following stocking into temperatures of 8, 6 and 4°C in a controlled laboratory environment. The southern population showed lower condition and higher mortality during early life than the northern population while increased rearing temperature impacted the growth and condition of developing northern sturgeon. During the cold shock, HSP70 and HSP90a mRNA expression increased in all sturgeon treatments as stocking temperature decreased, with higher expression observed in the southern population. Expression of HSP90b, CIRP and SCD increased as stocking temperature decreased in northern sturgeon with early acclimation to 20°C. Correlation analyses indicated the strongest molecular relationships were in the expression of HSP90b, CIRP and SCD, across all treatments, with a correlation between HSP90b and body condition in northern sturgeon with early acclimation to 20°C. Together, these observations highlight the importance of population and rearing environment throughout early development and on later cellular responses induced by cold stocking temperatures.

Endogenous cortisol production and its relationship with feeding transitions in larval lake sturgeon (Acipenser fulvescens).

Our understanding of the importance of cortisol in the development of fishes largely stems from teleosts and in particular the zebrafish, Danio rerio. However, studies examining the ontogeny of the cortisol endocrine axis in acipenseriformes (sturgeon and paddlefish) have demonstrated similar general patterns during early development. Beginning with maternal deposition of cortisol in the egg, followed by development of de novo synthesis, a hypo-responsive period, and finally the ability of the fish to appropriately increase whole-body levels of cortisol in response to a stressor. In the present study, we demonstrate a similar pattern of ontogeny in the cortisol response in lake sturgeon over two-year classes. Whole-body levels of cortisol were examined over two cohorts and found to be different in both concentration and timing of endogenous production. The 2016 cohort were found to have relatively high levels of cortisol and developed to first feeding approximately six days faster than the 2017 cohort with lower levels of cortisol. In the 2017 cohort, mRNA expression of steroidogenic acute regulatory protein (StAR) and glucocorticoid receptor 1 (GR1) increased just prior to the increase in cortisol and associated onset of exogenous feeding. Treatment in metyrapone, an inhibitor of 11ß-hydroxylase, significantly inhibited cortisol production and resulted in the inability of the fish to appropriately transition to exogenous feeding. Data suggest a potential key role for cortisol in lake sturgeon as they transition between diets during early life history.

Tissue-specific transcriptomes reveal potential mechanisms of microbiome heterogeneity in an ancient fish.

The lake sturgeon (Acipenser fulvescens) is an ancient, octoploid fish faced with conservation challenges across its range in North America, but a lack of genomic resources has hindered molecular research in the species. To support such research, we created a transcriptomic database from 13 tissues: brain, esophagus, gill, head kidney, heart, white muscle, liver, glandular stomach, muscular stomach, anterior intestine, pyloric cecum, spiral valve and rectum. The transcriptomes for each tissue were sequenced and assembled individually from a mean of 98.3 million (±38.9 million SD) reads each. In addition, an overall transcriptome was assembled and annotated with all data used for each tissue-specific transcriptome. All assembled transcriptomes and their annotations were made publicly available as a scientific resource. The non-gut transcriptomes provide important resources for many research avenues. However, we focused our analysis on messenger ribonucleic acid (mRNA) observations in the gut because the gut represents a compartmentalized organ system with compartmentalized functions, and seven of the sequenced tissues were from each of these portions. These gut-specific analyses were used to probe evidence of microbiome regulation by studying heterogeneity in microbial genes and genera identified from mRNA annotations. Gene set enrichment analyses were used to reveal the presence of photoperiod and circadian-related transcripts in the pyloric cecum, which may support periodicity in lake sturgeon digestion. Similar analyses were used to identify different types of innate immune regulation across the gut, while analyses of unique transcripts annotated to microbes revealed heterogeneous genera and genes among different gut tissues. The present results provide a scientific resource and information about the mechanisms of compartmentalized function across gut tissues in a phylogenetically ancient vertebrate. Database URL: https://figshare.com/projects/Lake_Sturgeon_Transcriptomes/133143.

Comparison of metabolic rate between two genetically distinct populations of lake sturgeon.

Environmental temperatures differ across latitudes in the temperate zone, with relatively lower summer and fall temperatures in the north leading to a shorter growing season prior to winter. As an adaptive response, during early life stages, fish in northern latitudes may grow faster than their conspecifics in southern latitudes, which potentially manifests as different allometric relationships between body mass and metabolic rate. In the present study, we examined if population or year class had an effect on the variation of metabolic rate and metabolic scaling of age-0 lake sturgeon (Acipenser fulvescens) by examining these traits in both a northern (Nelson River) and a southern (Winnipeg River) population. We compiled 6 years of data that used intermittent flow respirometry to measure metabolic rate within the first year of life for developing sturgeon that were raised in the same environment at 16°C. We then used a Bayesian modeling approach to examine the impacts of population and year class on metabolic rate and mass-scaling of metabolic rate. Despite previous reports of genetic differences between populations, our results showed that there were no significant differences in standard metabolic rate, routine metabolic rate, maximum metabolic rate, and metabolic scaling between the two geographically separated populations at a temperature of 16°C. Our analysis implied that the lack of metabolic differences between populations could be due to family effects/parental contribution, or the rearing temperature used in the study. The present research provided insights for conservation and reintroduction strategies for these populations of lake sturgeon, which are endangered or threatened across most of their natural range.

Non-Lethal Sampling Supports Integrative Movement Research in Freshwater Fish.

Freshwater ecosystems and fishes are enormous resources for human uses and biodiversity worldwide. However, anthropogenic climate change and factors such as dams and environmental contaminants threaten these freshwater systems. One way that researchers can address conservation issues in freshwater fishes is via integrative non-lethal movement research. We review different methods for studying movement, such as with acoustic telemetry. Methods for connecting movement and physiology are then reviewed, by using non-lethal tissue biopsies to assay environmental contaminants, isotope composition, protein metabolism, and gene expression. Methods for connecting movement and genetics are reviewed as well, such as by using population genetics or quantitative genetics and genome-wide association studies. We present further considerations for collecting molecular data, the ethical foundations of non-lethal sampling, integrative approaches to research, and management decisions. Ultimately, we argue that non-lethal sampling is effective for conducting integrative, movement-oriented research in freshwater fishes. This research has the potential for addressing critical issues in freshwater systems in the future.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Effects of acclimation temperature on the thermal physiology in two geographically distinct populations of lake sturgeon (Acipenser fulvescens).

Temperature is one of the most important abiotic factors regulating development and biological processes in ectotherms. By 2050, climate change may result in temperature increases of 2.1-3.4°C in Manitoba, Canada. Lake sturgeon, Acipenser fulvescens, from both northern and southern populations in Manitoba were acclimated to 16, 20 and 24°C for 30 days, after which critical thermal maximum (CTmax) trials were conducted to investigate their thermal plasticity. We also examined the effects of temperature on morphological and physiological indices. Acclimation temperature significantly influenced the CTmax, body mass, hepatosomatic index, metabolic rate and the mRNA expression of transcripts involved in the cellular response to heat shock and hypoxia (HSP70, HSP90a, HSP90b, HIF-1α) in the gill of lake sturgeon. Population significantly affected the above phenotypes, as well as the mRNA expression of Na+/K+ ATPase-α1 and the hepatic glutathione peroxidase enzyme activity. The southern population had an average CTmax that was 0.71 and 0.45°C higher than the northern population at 20 and 24°C, respectively. Immediately following CTmax trials, mRNA expression of HSP90a and HIF-1α was positively correlated with individual CTmax of lake sturgeon across acclimation treatments and populations (r = 0.7, r = 0.62, respectively; P < 0.0001). Lake sturgeon acclimated to 20 and 24°C had decreased hepatosomatic indices (93 and 244% reduction, respectively; P < 0.0001) and metabolic suppression (27.7 and 42.1% reduction, respectively; P < 0.05) when compared to sturgeon acclimated to 16°C, regardless of population. Glutathione peroxidase activity and mRNA expression Na+/K+ ATPase-α1 were elevated in the northern relative to the southern population. Acclimation to 24°C also induced mortality in both populations when compared to sturgeon acclimated to 16 and 20°C. Thus, increased temperatures have wide-ranging population-specific physiological consequences for lake sturgeon across biological levels of organization.