RNA-Seq Analysis of the Growth Hormone Transgenic Female Triploid Atlantic Salmon (Salmo salar) Hepatic Transcriptome Reveals Broad Temperature-Mediated Effects on Metabolism and Other Biological Processes.

This study examined the impact of rearing temperature (10.5, 13.5 or 16.5°C) on the hepatic transcriptome of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) at an average weight of 800 g. Six stranded PE libraries were Illumina-sequenced from each temperature group, resulting in an average of over 100 M raw reads per individual fish. RNA-sequencing (RNA-seq) results showed the greatest difference in the number of differentially expressed transcripts (1750 DETs), as revealed by both DESeq2 and edgeR (q < 0.05; fold-change > |1.5|), was between the 10.5 and 16.5°C temperature groups. In contrast, 172 and 52 DETs were found in the 10.5 vs. 13.5°C and the 13.5 vs. 16.5°C comparisons, respectively. Considering the DETs between the 10.5 and 16.5°C groups, 282 enriched gene ontology (GO) terms were identified (q < 0.05), including "response to stress", "immune system process", "lipid metabolic process", "oxidation-reduction process", and "cholesterol metabolic process", suggesting elevated temperature elicited broad effects on multiple biological systems. Pathway analysis using ClueGO showed additional impacts on amino acid and lipid metabolism. There was a significant positive correlation between RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) results for 8 of 9 metabolic-related transcripts tested. RT-qPCR results also correlated to changes in fillet tissue composition previously reported in these salmon (e.g., methionine and lysine concentrations positively correlated with hsp90ab1 transcript expression), suggesting that rearing temperature played a significant role in mediating metabolic/biosynthetic pathways of AquAdvantage Salmon. Many transcripts related to lipid/fatty acid metabolism (e.g., elovl2, fabpi, hacd2, mgll, s27a2, thrsp) were downregulated at 16.5°C compared to both other temperature groups. Additionally, enrichment of stress-, apoptosis- and catabolism-relevant GO terms at 16.5°C suggests that this temperature may not be ideal for commercial production when using freshwater recirculating aquaculture systems (RAS). This study relates phenotypic responses to transcript-specific findings and therefore aids in the determination of an optimal rearing temperature for AquAdvantage Salmon. With approval to grow and sell AquAdvantage Salmon in the United States and Canada, the novel insights provided by this research can help industry expansion by promoting optimal physiological performance and health.

Acute critical thermal maximum does not predict chronic incremental thermal maximum in Atlantic salmon (Salmo salar).

Atlantic salmon is an important aquaculture species farmed in ocean net-pens and therefore subjected to changing environmental conditions, including rising temperatures. This creates a need for research on the thermal tolerance of this species for the future of sustainable aquaculture. We investigated the thermal tolerance of individually tagged Atlantic salmon post-smolts subjected sequentially to two common high-temperature challenges: critical thermal maximum (CTmax) followed by incremental thermal maximum (ITmax). Our goals were (1) to determine whether CTmax can predict ITmax for individual fish, and (2) to examine connections between various body size (mass, length, condition factor), cardiac (absolute and relative ventricle mass) and blood (hematocrit) metrics and thermal tolerance. We found no relationship between CTmax and ITmax. This is of concern because CTmax, which is a quick and easy test, is often used to predict upper lethal limits in fish despite not using real-world rates of temperature increase and not using death as the experimental endpoint (unlike ITmax). Also, some metrics which correlated in one direction with CTmax had the opposite correlation with ITmax. For instance, smaller fish or fish with smaller ventricles had a higher CTmax but a lower ITmax than larger fish or fish with larger ventricles. Taken together, these results highlight the need to take care when using acute thermal tolerance tests to predict real-world responses to rising temperatures.

Acclimation to warmer temperature reversibly improves high-temperature hypoxia tolerance in both diploid and triploid brook charr, Salvelinus fontinalis.

Rising temperature leads to reduced oxygen solubility and therefore increases the risk of exposure to harmful hypoxic condition for fish in their natural aquatic environments and in aquaculture. The goal of this study was to determine whether acclimation to warmer temperature can improve high-temperature hypoxia tolerance in fish, using sibling diploid and triploid brook charr as the experimental model. Triploid fish are used for aquaculture and fisheries management because they are sterile, but they are known to have reduced thermal and hypoxia tolerance compared to conventional diploids. Fish were pre-acclimated to either 15 °C (optimum temperature for diploids) or 18 °C and then assessed for high-temperature hypoxia tolerance by rapidly increasing temperature to pre-determined levels (up to 30 °C), holding fish at these temperatures for one hour, and then using compressed nitrogen to drive oxygen out of the water. Hypoxia tolerance was expressed as both the oxygen tension at loss of equilibrium and the time taken to reach this endpoint following the start of the trial. Acclimation to 18 °C improved hypoxia tolerance at high temperatures but this advantage was lost after reacclimation to 15 °C. Although 18 °C acclimation improved the hypoxia tolerance of triploids, it remained inferior to that of diploids under identical test conditions. Somatic energy reserves (estimated as condition factor and hepatosomatic index), cardiac output (relative ventricular mass) and oxygen carrying capacity of the blood (hemoglobin concentration and hematocrit) did not markedly affect high-temperature hypoxia tolerance.

Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos.

Ploidy transitions through whole genome duplication have shaped evolution by allowing the sub- and neo-functionalization of redundant copies of highly conserved genes to express novel traits. The nuclear:cytoplasmic (n:c) ratio is maintained in polyploid vertebrates resulting in larger cells, but body size is maintained by a concomitant reduction in cell number. Ploidy can be manipulated easily in most teleosts, and the zebrafish, already well established as a model system for biomedical research, is therefore an excellent system in which to study the effects of increased cell size and reduced cell numbers in polyploids on development and physiology. Here we describe a novel technique using confocal microscopy to measure genome size and determine ploidy non-lethally at 48 h post-fertilization (hpf) in transgenic zebrafish expressing fluorescent histones. Volumetric analysis of myofiber nuclei using open-source software can reliably distinguish diploids and triploids from a mixed-ploidy pool of embryos for subsequent experimentation. We present an example of this by comparing heart rate between confirmed diploid and triploid embryos at 54 hpf.

Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos.

Hypoxia induces precocious hatching in zebrafish, but we do not have a clear understanding of the molecular mechanisms regulating the activation of the hatching enzyme or how these mechanisms trigger precocious hatching under unfavorable environmental conditions. Using immunohistochemistry, pharmacological inhibition of matrix metalloproteinase 13 (Mmp13), and in vivo zymography, we show that Mmp13a is present in the hatching gland just as embryos become hatching competent and that Mmp13a activity is required for both normal hatching and hypoxia-induced precocious hatching. We conclude that Mmp13a likely functions in activating the hatching enzyme zymogen and that Mmp13a activity is necessary but not sufficient for hatching in zebrafish. This study highlights the broad nature of MMP function in development and provides a non-mammalian example of extra-embryonic processes mediated by MMP activity.

Impact of rearing temperature on the innate antiviral immune response of growth hormone transgenic female triploid Atlantic salmon (Salmo salar).

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.

Ploidy Has Minimal Effect on Hypoxia Tolerance at High Temperature in Rainbow Trout (Oncorhynchus mykiss).

Polyploidy is an important driver of evolutionary change (generally via tetraploidy) and also serves a practical role in aquaculture and fisheries management (via triploidy). Fundamental changes in cell size and number that accompany polyploidy are predicted to affect cellular and whole-animal physiology due to constraints placed on surface-mediated processes at the cellular level, potentially altering environmental tolerances and optima. The aim of this study was to determine whether the documented reduction in thermal tolerance of aquatic polyploids is a result of their being less hypoxia tolerant. This was assessed by holding diploid and triploid rainbow trout for 1 h above their thermal optima in separate trials at eight temperatures between 20° and 27°C and then rapidly reducing the oxygen tension (Po2) of the water and determining the nonlethal Po2 at which fish lost equilibrium. As expected, there was a highly significant ([Formula: see text]) effect of temperature on Po2 at loss of equilibrium. Although there was also a significant ([Formula: see text]) effect of ploidy on Po2 at loss of equilibrium, with triploid values higher than diploid, post hoc analyses showed no significant effect of ploidy at any specific temperature. Oxygen availability alone therefore does not appear to play a major role in determining the thermal tolerance of polyploids.

Erythrocyte heat shock protein responses to chronic (in vivo) and acute (in vitro) temperature challenge in diploid and triploid salmonids.

This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions.

The effects of dissolved oxygen and triploidy on critical thermal maximum in brook charr (Salvelinus fontinalis).

This study examined the effect of dissolved oxygen (DO) level on critical thermal maximum (CTMax) in diploid and triploid brook charr (Salvelinus fontinalis) exposed to a temperature increase of 3°C/h. Because gas solubility is inversely proportional to temperature, DO declines during standard CTMax tests. With this treatment as a baseline, oxygen or nitrogen injection was used to provide three other DO conditions during CTMax tests: two hyperoxic (maintenance at initial 10mg/L and increase from 10mg/L at 2mg/L/h) and one hypoxic (decrease from 10mg/L at 2mg/L/h). Hyperoxia had no effect on temperature at CTMax or time taken to reach CTMax. Hypoxia, on the other hand, resulted in a significantly lower CTMax and shorter time to CTMax than under standard or hyperoxic conditions, with both indices affected by triploidy but not in a consistent fashion: in one experiment triploids had a lower CTMax and shorter time to CTMax than diploids and in a second experiment they had a higher CTMax and longer time to CTMax than diploids. Indices of the secondary stress response (plasma glucose and ions) during CTMax tests under hypoxia responded as would be predicted for an acute stress, with no difference between triploids and diploids.

Optimal swimming speeds reflect preferred swimming speeds of brook charr (Salvelinus fontinalis Mitchill, 1874).

Several measures have been developed to quantify swimming performance to understand various aspects of ecology and behaviour, as well as to help design functional applications for fishways and aquaculture. One of those measures, the optimal swimming speed, is the speed at which the cost of transport (COT) is minimal, where COT is defined as the cost of moving unit mass over unit distance. The experimental protocol to determine the optimal swimming speed involves forced-swimming in a flume or respirometer. In this study, a 4.5-m-long tilted raceway with gradually increasing upstream water speed is used to determine a novel, behaviourally based swimming parameter: the preferred swimming speed. The optimal swimming speed and the preferred swimming speed of brook charr were determined and a comparison of the two reveals that the optimal swimming speed (25.9 ± 4.5 cm s⁻¹ or 1.02 ± 0.47 bl s⁻¹) reflected the preferred swimming speed (between 20 cm s⁻¹ or 0.78 ± 0.02 bl s⁻¹ and 25 cm s⁻¹ or 0.95 ± 0.03 bl s⁻¹). The preferred swimming speed can be advantageous for the determination of swimming speeds for the use in aquaculture studies.

Effect of acclimation temperature on routine metabolic rate in triploid salmonids.

The objective of this research was to determine whether triploid fish differ from diploids in their routine metabolic rates across a range of acclimation temperatures. Sibling diploids and triploids were acclimated to 12, 15 and 18 degrees C (Atlantic salmon; Salmo salar) and to 9, 12 and 15 degrees C (brook charr; Salvelinus fontinalis) prior to experimentation. Routine metabolic rates were then determined three times over a two-month period. Triploids of both species had higher metabolic rates than diploids at lower temperatures, and lower metabolic rates than diploids at higher temperatures, demonstrating that triploids have different (i.e., lower) thermal optima than diploids. This likely explains prior observations of high mortality of triploids at chronically elevated, but sub-lethal, rearing temperatures for sibling diploids.

Gonadal differentiation and hormonal sex reversal in Arctic charr (Salvelinus alpinus).

The purpose of this study was to develop a practical protocol for the production of female populations of Arctic charr (Salvelinus alpinus). Achieving this required knowledge of the timing of gonadal differentiation. Undifferentiated gonads were observed microscopically to be present by 194 degrees C-days post-hatch and definitive germ cells by 346 degrees C-days post-hatch, where " degrees C-days" denote acquired thermal units calculated as the product of temperature and days. Some of the gonads had developed a lumen by 510 degrees C-days post-hatch, and by 681 degrees C-days post-hatch anatomical divergence into two types of gonads was clear. Two protocols (immersion and feeding) were tested for hormonal sex reversal of genotypic females using the synthetic androgen 17alpha-methyldihydrotestosterone (MDHT). Six-hour MDHT immersions (0.5, 1, 3, 5 and 10 mg/L) were carried out weekly from hatch to first feeding (140 degrees C-days post-hatch), whereas daily feeding treatments (0.5 mg/kg) went from 140 to 600 degrees C-days post-hatch. The sex ratios of all immersion experimental groups were significantly different from the control, with the proportion of presumptive males increasing as MDHT concentration increased. The highest immersion treatment, 10 mg/L, yielded a population of 90% presumptive males and 10% with atypical gonads. However, the most effective treatment, yielding a population of 90% presumptive males and no fish with atypical gonads, was the feeding treatment. Given that female salmonid fishes are homogametic, sex-reversed (masculinized) genotypic females produced in this way can serve as broodstock for the creation of all-female charr populations for aquaculture.

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development.

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.