Atlantic Salmon (Salmo salar) bacterial and viral innate immune responses are not impaired by florfenicol or tetracycline administration.

Antibiotics are used to treat bacterial infections in fish aquaculture, and these drugs can interact with immune cells/the immune system and potentially leave fish vulnerable to viral, fungal, parasitic, or other bacterial infections. However, the effects of antibiotics on fish immunity have largely been overlooked by the aquaculture industry. We tested, at 12 and 20 °C, whether tetracycline and florfenicol (the most commonly used antibiotics in commercial aquaculture), affected the Atlantic salmon's capacity to respond to bacterial or viral stimulation. Atlantic salmon were acclimated to 12 or 20 °C and fed with tetracycline or florfenicol (100 and 10 mg kg of body weight-1 day-1, respectively) medicated feed for 15 or 10 days, respectively. Thereafter, we evaluated their immune function prior to, and after, an intraperitoneal injection of Forte Micro (containing inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC). We measured the transcript expression levels of 8 anti-bacterial and 8 anti-viral putative biomarker genes, and the innate (leukocyte respiratory burst, plasma lysozyme activity and hemolytic activity of the alternative complement pathway) and cellular (relative number of erythrocytes, lymphocytes and thrombocytes, and granulocytes such as monocytes and neutrophils) responses to these challenges. Overall, we only found a few minor effects of either tetracycline or florfenicol on immune gene expression or function at either temperature. Although several studies have reported that antibiotics may negatively affect fish immune responses, our results show that industry-relevant dietary tetracycline and florfenicol treatments do not substantially impact the salmon's innate immune responses. Currently, this is the most comprehensive study on the effects of antibiotics administrated according to industry protocols on immune function in Atlantic salmon.

Comparative Genomics of Typical and Atypical Aeromonas salmonicida Complete Genomes Revealed New Insights into Pathogenesis Evolution.

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies' taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Atlantic Salmon (Salmo salar) Cage-Site Distribution, Behavior, and Physiology During a Newfoundland Heat Wave.

Background: Climate change is leading to increased water temperatures and reduced oxygen levels at sea-cage sites, and this is a challenge that the Atlantic salmon aquaculture industry must adapt to it if it needs to grow sustainably. However, to do this, the industry must better understand how sea-cage conditions influence the physiology and behavior of the fish. Method: We fitted ~2.5 kg Atlantic salmon on the south coast of Newfoundland with Star-Oddi milli-HRT ACT and Milli-TD data loggers (data storage tags, DSTs) in the summer of 2019 that allowed us to simultaneously record the fish's 3D acceleration (i.e., activity/behavior), electrocardiograms (and thus, heart rate and heart rate variability), depth, and temperature from early July to mid-October. Results: Over the course of the summer/fall, surface water temperatures went from ~10-12 to 18-19.5°C, and then fell to 8°C. The data provide valuable information on how cage-site conditions affected the salmon and their determining factors. For example, although the fish typically selected a temperature of 14-18°C when available (i.e., this is their preferred temperature in culture), and thus were found deeper in the cage as surface water temperatures peaked, they continued to use the full range of depths available during the warmest part of the summer. The depth occupied by the fish and heart rate were greater during the day, but the latter effect was not temperature-related. Finally, while the fish generally swam at 0.4-1.0 body lengths per second (25-60 cm s-1), their activity and the proportion of time spent using non-steady swimming (i.e., burst-and-coast swimming) increased when feeding was stopped at high temperatures. Conclusion: Data storage tags that record multiple parameters are an effective tool to understand how cage-site conditions and management influence salmon (fish) behavior, physiology, and welfare in culture, and can even be used to provide fine-scale mapping of environmental conditions. The data collected here, and that in recent publications, strongly suggest that pathogen (biotic) challenges in combination with high temperatures, not high temperatures + moderate hypoxia (~70% air saturation) by themselves, are the biggest climate-related challenge facing the salmon aquaculture industry outside of Tasmania.

The Relationship between Myoglobin, Aerobic Capacity, Nitric Oxide Synthase Activity and Mitochondrial Function in Fish Hearts.

The dynamic interactions between nitric oxide (NO) and myoglobin (Mb) in the cardiovascular system have received considerable attention. The loss of Mb, the principal O2 carrier and a NO scavenger/producer, in the heart of some red-blooded fishes provides a unique opportunity for assessing this globin's role in NO homeostasis and mitochondrial function. We measured Mb content, activities of enzymes of NO and aerobic metabolism [NO Synthase (NOS) and citrate synthase, respectively] and mitochondrial parameters [Complex-I and -I+II respiration, coupling efficiency, reactive oxygen species production/release rates and mitochondrial sensitivity to inhibition by NO (i.e., NO IC50)] in the heart of three species of red-blooded fish. The expression of Mb correlated positively with NOS activity and NO IC50, with low NOS activity and a reduced NO IC50 in the Mb-lacking lumpfish (Cyclopterus lumpus) as compared to the Mb-expressing Atlantic salmon (Salmo salar) and short-horned sculpin (Myoxocephalus scorpius). Collectively, our data show that NO levels are fine-tuned so that NO homeostasis and mitochondrial function are preserved; indicate that compensatory mechanisms are in place to tightly regulate [NO] and mitochondrial function in a species without Mb; and strongly suggest that the NO IC50 for oxidative phosphorylation is closely related to a fish's hypoxia tolerance.

Acclimation to warm temperatures has important implications for mitochondrial function in Atlantic salmon (Salmo salar).

In fish, the capacity of thermal acclimation to preserve cardiac mitochondrial function under future warming scenarios is important to understand given the central roles that cardiac energy metabolism and performance play in this taxa's thermal tolerance. We acclimated Atlantic salmon to 12 and 20°C (for >2 months), and investigated the effects of acute and chronic warming on cardiac mitochondrial respiration and reactive oxygen species (ROS) production (release rate) using high-resolution fluorespirometry. Further, we compared the sensitivity of mitochondrial respiration to nitric oxide (i.e. the NO IC50), and assessed the mitochondrial response to anoxia-reoxygenation (AR). Acute exposure to 20°C increased maximal mitochondrial respiration by ∼55%; however, the mitochondria's complex I respiratory control ratio was 17% lower and ROS production was increased by ≥60%. Acclimation to 20°C: (1) preserved mitochondrial coupling and aerobic capacity; (2) decreased the mitochondria's ROS production by ∼30%; (3) increased the mitochondria's NO IC50 by ∼23%; and (4) improved mitochondrial membrane integrity at 20°C. AR did not affect mitochondrial function at 12°C, but acute exposure to 20°C and AR depressed maximal mitochondrial respiration (by ∼9%) and coupling (by ∼16%) without impacting ROS production. Finally, warm acclimation did not improve the capacity of mitochondria to recover from AR, indicating that there was no 'cross-tolerance' between these challenges. Our findings provide compelling evidence that thermal plasticity of cardiac mitochondrial function contributes to the Atlantic salmon's capability to survive at ≥20°C for prolonged periods, but call into question whether this plasticity may allow them to withstand high temperatures when combined with other stressors.

Improved mitochondrial function in salmon (Salmo salar) following high temperature acclimation suggests that there are cracks in the proverbial 'ceiling'.

Mitochondrial function can provide key insights into how fish will respond to climate change, due to its important role in heart performance, energy metabolism and oxidative stress. However, whether warm acclimation can maintain or improve the energetic status of the fish heart when exposed to short-term heat stress is not well understood. We acclimated Atlantic salmon, a highly aerobic eurythermal species, to 12 and 20 °C, then measured cardiac mitochondrial functionality and integrity at 20 °C and at 24, 26 and 28 °C (this species' critical thermal maximum ± 2 °C). Acclimation to 20 °C vs. 12 °C enhanced many aspects of mitochondrial respiratory capacity and efficiency up to 24 °C, and preserved outer mitochondrial membrane integrity up to 26 °C. Further, reactive oxygen species (ROS) production was dramatically decreased at all temperatures. These data suggest that salmon acclimated to 'normal' maximum summer temperatures are capable of surviving all but the most extreme ocean heat waves, and that there is no 'tradeoff' in heart mitochondrial function when Atlantic salmon are acclimated to high temperatures (i.e., increased oxidative phosphorylation does not result in heightened ROS production). This study suggests that fish species may show quite different acclimatory responses when exposed to prolonged high temperatures, and thus, susceptibility to climate warming.

The Innate Immune Response of Atlantic Salmon (Salmo salar) Is Not Negatively Affected by High Temperature and Moderate Hypoxia.

Climate change is predicted to increase water temperatures and decrease oxygen levels in freshwater and marine environments, however, there is conflicting information regarding the extent to which these conditions may impact the immune defenses of fish. In this study, Atlantic salmon were exposed to: (1) normoxia (100-110% air saturation) at 12°C; (2) an incremental temperature increase (1°C per week from 12 to 20°C), and then held at 20°C for an additional 4 weeks; and (3) "2" with the addition of moderate hypoxia (~65-75% air saturation). These conditions realistically reflect what farmed salmon in some locations are currently facing, and future conditions in Atlantic Canada and Europe, during the summer months. The salmon were sampled for the measurement of head kidney constitutive anti-bacterial and anti-viral transcript expression levels, and blood parameters of humoral immune function. Thereafter, they were injected with either the multi-valent vaccine Forte V II (contains both bacterial and viral antigens) or PBS (phosphate-buffer-saline), and the head kidney and blood of these fish were sampled at 6, 12, 24, and 48 h post-injection (HPI). Our results showed that: (1) neither high temperature, nor high temperature + moderate hypoxia, adversely affected respiratory burst, complement activity or lysozyme concentration; (2) the constitutive transcript expression levels of the anti-bacterial genes il1ß, il8-a, cox2, hamp-a, stlr5-a, and irf7-b were up-regulated by high temperature; (3) while high temperature hastened the peak in transcript expression levels of most anti-bacterial genes by 6-12 h following V II injection, it did not affect the magnitude of changes in transcript expression; (4) anti-viral (viperin-b, mx-b, and isg15-a) transcript expression levels were either unaffected, or downregulated, by acclimation temperature or V II injection over the 48 HPI; and (5) hypoxia, in addition to high temperature, did not impact immune transcript expression. In conclusion, temperatures up to 20°C, and moderate hypoxia, do not impair the capacity of the Atlantic salmon's innate immune system to respond to bacterial antigens. These findings are surprising, and highlight the salmon's capacity to mount robust innate immune responses (i.e., similar to control fish under optimal conditions) under conditions approaching their upper thermal limit.

Cardiac mitochondrial function, nitric oxide sensitivity and lipid composition following hypoxia acclimation in sablefish.

In fishes, the effect of O2 limitation on cardiac mitochondrial function remains largely unexplored. The sablefish (Anoplopoma fimbria) encounters considerable variations in environmental oxygen availability, and is an interesting model for studying the effects of hypoxia on fish cardiorespiratory function. We investigated how in vivo hypoxia acclimation (6 months at 40% then 3 weeks at 20% air saturation) and in vitro anoxia-reoxygenation affected sablefish cardiac mitochondrial respiration and reactive oxygen species (ROS) release rates using high-resolution fluorespirometry. Further, we investigated how hypoxia acclimation affected the sensitivity of mitochondrial respiration to nitric oxide (NO), and compared mitochondrial lipid and fatty acid (FA) composition between groups. Hypoxia acclimation did not alter mitochondrial coupled or uncoupled respiration, or respiratory control ratio, ROS release rates, P50 or superoxide dismutase activity. However, it increased citrate synthase activity (by ∼20%), increased the sensitivity of mitochondrial respiration to NO inhibition (i.e., the NO IC50 was 25% lower), and enhanced the recovery of respiration (by 21%) and reduced ROS release rates (by 25-30%) post-anoxia. In addition, hypoxia acclimation altered mitochondrial FA composition [increasing arachidonic acid (20:4ω6) and eicosapentaenoic acid (20:5ω3) proportions by 11 and 14%, respectively], and SIMPER analysis revealed that the phospholipid:sterol ratio was the largest contributor (24%) to the dissimilarity between treatments. Overall, these results suggest that hypoxia acclimation may protect sablefish cardiac bioenergetic function during or after periods of O2 limitation, and that this may be related to alterations in mitochondrial sensitivity to NO and to adaptive changes in membrane composition (fluidity).

The acute and incremental thermal tolerance of Atlantic cod (Gadus morhua) families under normoxia and mild hypoxia.

Given climate change projections, the limited ability of fish reared in sea-cages to behaviourally thermoregulate, and that thermal tolerance may be heritable, studies that examine family-related differences in upper thermal tolerance are quite relevant to the aquaculture industry. Thus, we investigated the upper thermal tolerance of 15 Atlantic cod (Gadus morhua L.) families by challenging them with acute (2 °C h-1) and incremental (1 °C every 4 days) temperature increases (CTmax and ITmax tests, respectively) under normoxia (~ 100% air saturation) and mild hypoxia (~ 75% air sat.). The cod's CTmax was 22.5 ±â€¯0.1 °C (mean ±â€¯S.E.) during normoxia and 21.8 ±â€¯0.1 °C during hypoxia (P < 0.001); and these two CTmax values were significantly correlated across families. In both the normoxic and hypoxic ITmax tests, feed intake fell by ~50% between 17 and 18 °C, and stopped entirely by 21 °C. No mortalities were observed under 20 °C in the normoxic and hypoxic ITmax tests, and the ITmax value was ~21.7 °C in both groups. Differences in the upper thermal tolerance between families were only observed in the CTmax experiment. No correlation was found between the specific growth rate and the CTmax of the families. Further, no correlation existed between CTmax and ITmax. This study is the first to compare the thermal tolerance of fish families to both CTmax and ITmax challenges, and the data: 1) suggest that the Atlantic cod is quite tolerant of acute (i.e., hours) or short-term (i.e., weeks) exposure to high water temperatures (i.e., up to 20 °C); 2) indicate that it might be difficult to select fish with higher ITmax values; and 3) question the relevance of CTmax for selecting fish that are destined for sea-cages where temperatures slowly warm over the summer.

The environmental tolerances and metabolic physiology of sablefish (Anoplopoma fimbria).

Given the potential impacts of global warming, such as increases in temperature and the frequency/severity of hypoxia in marine ecosystems, it is important to study the impacts of these environmental challenges on sea-cage reared aquaculture species. This study focuses on the sablefish (Anoplopoma fimbria), an emerging aquaculture species that has a unique ecology in the wild. For instance, adults inhabit oxygen minimum zones and cool waters at depths up to 1500 m. Using Atlantic salmon (Salmo salar) (~1132 g adults) as a comparative species, we used intermittent-flow respirometry to characterize the tolerance and metabolic response of sablefish (~10 g juveniles and ~675 g adults) to acute increases in temperature (2 °C h-1) and decreases in oxygen level (~10% air saturation h-1). Adult sablefish were much more hypoxia tolerant than adult salmon [O2 level at loss of equilibrium ~5.4% vs. ~24.2% air saturation, respectively]. In addition, sablefish could withstand upper temperatures only slightly lower than salmon [critical thermal maximum (CTmax) ~24.9 °C vs. ~26.2 °C, respectively]. Sablefish juveniles were both less hypoxia and thermally tolerant than adults [critical O2 tension ~18.9% vs. ~15.8% air saturation; CTmax ~22.7 vs. ~24.9 °C, respectively]. Interestingly, many of these differences in environmental tolerance could not be explained by differences in metabolic parameters (aerobic scope or routine metabolic rate). Our findings show that sablefish are tolerant of high temperatures, and very tolerant of hypoxia, traits that are advantageous for an aquaculture species in the era of climate change.