A parasite outbreak in notothenioid fish in an Antarctic fjord.

Climate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystems.

A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity.

Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 µg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3+CD4+ and CD3+CD8+ T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants.

Spotted Wolffish Broodstock Management and Egg Production: Retrospective, Current Status, and Research Priorities.

The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in the mid-1990s as this species was considered by Norwegian authorities to be a top candidate species for cold-water aquaculture in the North Atlantic regions. Previous research conducted in Norway (since 1992) and Canada (since 2000), focused on identifying key biological parameters for spotted wolffish cultivation which led, respectively, to the rapid establishment of a full commercial production line in northern Norway, while Québec (Canada) is witnessing its first privately driven initiative to establish commercial production of spotted wolffish on its territory. The control of reproduction can be viewed as a major requirement to achieve the development of performant strains using genetic selection tools and/or all-year-round production to bring about maximal productivity and synchronization among a given captive population. Although the basic reproduction aspects are more understood and controlled there are still some challenges remaining involving broodstock and upscaling of operations that limit the achievement of a standardized production at the commercial level. Quality of gametes is still considered a major constraint and it can be affected by multiple factors including nutrition, environmental conditions, handling practices, and welfare status. Internal insemination/fertilization and the protracted incubation period are challenging as well as the establishment of a health monitoring program to secure large-scale operations. The profound progress achieved in the control of reproduction, sperm handling, and cryopreservation methods for this species is presented and discussed. In this review, we also go into detail over the full range of up-to-date cultivation practices involving broodstock and identify areas that could benefit from additional research efforts (i.e., broodstock nutrition, health and welfare, scaling-up egg and larval production, genetics, and development of selective breeding programs).

Isotopic Discrimination (δ15N, δ13C) in Captive and Wild Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica).

Studying the diet of consumers using stable isotopes provides insight into the foraging ecology of individuals and species. To accurately reconstruct the integrated diet of animals using stable isotope values, we must quantify diet-tissue discrimination factors (DTDFs), or the way in which stable isotopes in prey are incorporated into the tissues of consumers. To quantify DTDFs, controlled experiments are needed, whereby consumers are fed a constant diet. However, relatively few controlled-diet studies have been conducted for seabirds. In this study, captive adult Atlantic puffins (Fratercula arctica) and common murres (Uria aalge) were fed a two-source diet of capelin (Mallotus villosus) and Atlantic silverside (Menidia menidia) to determine the DTDFs for the cellular component of blood and plasma for both δ15N and δ13C. The DTDFs for the cellular component (Δ15N: 2.80±0.28; Δ13C: 1.21±0.22) and plasma (Δ15N: 1.72±1.03; Δ13C: -0.18±0.56) of puffins were similar to those for the cellular component (Δ15N: 2.91±0.18; Δ13C: 1.09±0.23) and plasma (Δ15N: 2.18±0.77; Δ13C: -0.70±0.18) of murres. We reconstructed the diet of wild murres and puffins breeding on the northeastern coast of Newfoundland using previously published DTDFs and estimated DTDFs from our feeding experiment. Reconstructed dietary proportions supported a priori knowledge of diet, although outputs were sensitive to the DTDF used. Despite the similarity of our DTDFs for puffins and murres, along with the similarity of our DTDFs with those of other seabird species, our sensitivity analysis revealed considerable differences among resultant dietary contributions from mixing models, further highlighting the importance of using species- and tissue-specific DTDFs to enhance knowledge in the foraging ecology of seabirds using stable isotopes.

Thermal tolerance and fish heart integrity: fatty acids profiles as predictors of species resilience.

The cardiovascular system is a major limiting system in thermal adaptation, but the exact physiological mechanisms underlying responses to thermal stress are still not completely understood. Recent studies have uncovered the possible role of reactive oxygen species production rates of heart mitochondria in determining species' upper thermal limits. The present study examines the relationship between individual response to a thermal challenge test (CTmax), susceptibility to peroxidation of membrane lipids, heart fatty acid profiles and cardiac antioxidant enzyme activities in two salmonid species from different thermal habitats (Salvelinus alpinus, Salvelinus fontinalis) and their hybrids. The susceptibility to peroxidation of membranes in the heart was negatively correlated with individual thermal tolerance. The same relationship was found for arachidonic and eicosapentaenoic acid. Total H2O2 buffering activity of the heart muscle was higher for the group with high thermal resistance. These findings underline a potential general causative relationship between sensitivity to oxidative stress, specific fatty acids, antioxidant activity in the cardiac muscle and thermal tolerance in fish and likely other ectotherms. Heart fatty acid profile could be indicative of species resilience to global change, and more importantly the plasticity of this trait could predict the adaptability of fish species or populations to changes in environmental temperature.

From Africa to Antarctica: Exploring the Metabolism of Fish Heart Mitochondria Across a Wide Thermal Range.

The thermal sensitivity of ectotherms is largely dictated by the impact of temperature on cellular bioenergetics, particularly on mitochondrial functions. As the thermal sensitivity of bioenergetic pathways depends on the structural and kinetic properties of its component enzymes, optimization of their collective function to different thermal niches is expected to have occurred through selection. In the present study, we sought to characterize mitochondrial phenotypic adjustments to thermal niches in eight ray-finned fish species occupying a wide range of thermal habitats by comparing the activities of key mitochondrial enzymes in their hearts. We measured the activity of four enzymes that control substrate entrance into the tricarboxylic acid (TCA) cycle: pyruvate kinase (PK), pyruvate dehydrogenase complex (PDHc), carnitine palmitoyltransferase (CPT), and hydroxyacyl-CoA dehydrogenase (HOAD). We also assayed enzymes of the electron transport system (ETS): complexes I, II, I + III, and IV. Enzymes were assayed at five temperatures (5, 10, 15, 20, and 25°C). Our results showed that the activity of CPT, a gatekeeper of the fatty acid pathway, was higher in the cold-water fish than in the warmer-adapted fish relative to the ETS (complexes I and III) when measured close to the species optimal temperatures. The activity of HOAD showed a similar pattern relative to CI + III and thermal environment. By contrast, PDHc and PK did not show the similar patterns with respect to CI + III and temperature. Cold-adapted species had high CIV activities compared to those of upstream complexes (I, II, I + III) whereas the converse was true for warm-adapted species. Our findings reveal a significant variability of heart mitochondrial organization among species that can be linked to temperature adaptation. Cold-adapted fish do not appear to compensate for PDHc activity but likely adjust fatty acids oxidation through higher activities of CPT and HOAD relative to complexes I + III.

Intergeneric hybrids inform reproductive isolating barriers in the Antarctic icefish radiation.

Interspecific hybridization or barriers to hybridization may have contributed to the diversification of Antarctic icefishes (Channichthyidae), but data supporting these hypotheses is scarce. To understand the potential for hybridization and to investigate reproductive isolating mechanisms among icefish species, we performed in vitro fertilization experiments using eggs from a female blackfin icefish Chaenocephalus aceratus and sperm from a male of another genera, the ocellated icefish Chionodraco rastrospinosus. Sequencing of genomic and mitochondrial DNA confirmed the intergeneric hybrid nature of resulting embryos which successfully developed and hatched as active larvae at about four and a half months during the Antarctic winter. This result demonstrates the compatibility of gametes of these two species and the viability of resulting zygotes and larvae. Due to logistic constraints and the slow developmental rate of icefishes, we could not test for long-term hybrid viability, fertility, fitness, or hybrid breakdown. Analysis of our fishing records and available literature, however, suggests that the strongest barriers to hybridization among parapatric icefish species are likely to be behavioral and characterized by assortative mating and species-specific courtship and nesting behaviors. This conclusion suggests that, in long-lived fish species with late sexual maturity and high energetic investment in reproduction like icefishes, pre-mating barriers are energetically more efficient than post-mating barriers to prevent hybridization.

Hybridization between char species (Salvelinus alpinus and Salvelinus fontinalis): a fast track for novel allometric trajectories.

Hybridization between closely related species can generate genetic and phenotypic variation, providing valuable biological material to assess the physiological impact of the structural or functional variability of different organs. In the present study, we examined growth rates of various organs and whole body in brook char, Arctic char and their reciprocal hybrids over a period of 281 days. Parental species achieved significantly higher body mass than their hybrids. Hybridization significantly reduced the relative size of the heart, liver and spleen. The relative size of pyloric caeca did not differ among the four groups. The observed lower growth performance of the hybrids compared to parental species strongly suggests that divergence in the relative size of digestive organs, liver and heart partly dictate growth capacity. Our results also suggest that the increased variability achieved through hybridization may prove useful in a genetic selection program.

Effects of fasting and refeeding on protein and glucose metabolism in Arctic charr.

Refeeding, following a period of food deprivation will often lead to compensatory growth. Although many studies have focused on molecular mechanisms behind this accelerated growth response in fish, little is known on the roles of protein and metabolism. We also assessed, for the first time, the potential roles of miRNAs in regulating compensatory growth. Artcic charr, Salvelinus alpinus, a northern freshwater species, was deprived of food for 101 days and then fed to satiety for 126 days. The refeeding period resulted in compensatory growth, with a partial compensation of body mass. The feed deprivation period lead to a decrease in hepatosomatic index (HSI) and intestinal somatic index (ISI). HSI and ISI were then gradually replenished during early refeeding, following a lag phase prior to the compensatory growth response. mRNA transcripts regulating protein degradation via the autophagy pathway (Cathepsin D and Cathepsin L) in muscle were upregulated during feed restriction and downregulated after refeeding, which could allow for greater protein accretion in muscle, facilitating compensatory growth. Transcript levels from the ubiquitin proteasome pathway (Mafbx and Murf1) and the calpain system (Calpain 7 and Calpastatin) suggested that these pathways were not involved in regulating compensatory growth. Furthermore, we've shown that miRNAs (miR-29a and miR-223) could be involved in fish glycogen homeostasis during the early stages of refeeding. These findings provide a deeper understanding of the molecular mechanisms regulating growth in fish.

Thermal tolerance and thermal sensitivity of heart mitochondria: Mitochondrial integrity and ROS production.

Cardiac mitochondrial metabolism provides 90% of the ATP necessary for the contractile exertion of the heart muscle. Mitochondria are therefore assumed to play a pivotal role in heart failure (HF), cardiovascular disease and ageing. Heat stress increases energy metabolism and oxygen demand in tissues throughout the body and imposes a major challenge on the heart, which is suspected of being the first organ to fail during heat stress. The underlying mechanisms inducing heart failure are still unclear. To pinpoint the processes implicated in HF during heat stress, we measured mitochondrial respiration rates and hydrogen peroxide production of isolated Arctic char (Salvelinus alpinus) heart mitochondria at 4 temperatures: 10°C (acclimation), 15°C, 20°C and 25°C (just over critical maximum). We found that at temperature ranges causing the loss of an organism's general homeostasis (between 20°C and 25°C) and with a substrate combination close to physiological conditions, the heat-induced increase in mitochondrial oxygen consumption levels off. More importantly, at the same state, hydrogen peroxide efflux increased by almost 50%. In addition, we found that individuals with low mitochondrial respiration rates produced more hydrogen peroxide at 10°C, 15°C and 20°C. This could indicate that individuals with cardiac mitochondria having a low respiratory capacity, have a more fragile heart and will be more prone to oxidative stress and HF, and less tolerant to temperature changes and other stressors. Our results show that, at temperatures close to the thermal limit, mitochondrial capacity is compromised and ROS production rates increase. This could potentially alter the performance of the cardiac muscle and lead to heat-induced HF underlining the important role that mitochondria play in setting thermal tolerance limits.

Embryogenesis and early skeletogenesis in the antarctic bullhead notothen, Notothenia coriiceps.

BACKGROUND: Environmental temperature influences rates of embryonic development, but a detailed staging series for vertebrate embryos developing in the subzero cold of Antarctic waters is not yet available from fertilization to hatching. Given projected warming of the Southern Ocean, it is imperative to establish a baseline to evaluate potential effects of changing climate on fish developmental dynamics. RESULTS: We studied the Bullhead notothen (Notothenia coriiceps), a notothenioid fish inhabiting waters between -1.9 and +2 °C. In vitro fertilization produced embryos that progressed through cleavage, epiboly, gastrulation, segmentation, organogenesis, and hatching. We compared morphogenesis spatially and temporally to Zebrafish and medaka. Experimental animals hatched after about 6 months to early larval stages. To help understand skeletogenesis, we analyzed late embryos for expression of sox9 and runx2, which regulate chondrogenesis, osteogenesis, and eye development. Results revealed that, despite their prolonged developmental time course, N. coriiceps embryos developed similarly to those of other teleosts with large yolk cells. CONCLUSIONS: Our studies set the stage for future molecular analyses of development in these extremophile fish. Results provide a foundation for understanding the impact of ocean warming on embryonic development and larval recruitment of notothenioid fish, which are key factors in the marine trophic system. Developmental Dynamics 245:1066-1080, 2016. © 2016 Wiley Periodicals, Inc.

Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity.

Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl2/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2-787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.

Ontogenetic effects of diet during early development on growth performance, myosin mRNA expression and metabolic enzyme activity in Atlantic cod juveniles reared at different salinities.

This study investigates the effect of diet during early development on growth and metabolic capacity in the juvenile stage of Atlantic cod. Growth in three groups of Atlantic cod juveniles (10-70 g) was measured at two salinities (15 per thousand or 32 per thousand) in combination with two temperatures (10 degrees C or 14 degrees C). Groups of cod from a single egg batch differed by having been fed with rotifers (R) or natural zooplankton (Z) during the first 36 days post hatch. A third group was fed zooplankton from 1 to 22 dph, after which diet changed to rotifers from 22 to 36 dph (ZRZ). All fish were weaned at 36 dph. Juveniles from the Z and ZRZ groups performed equally well under all experimental conditions, but fish that had received rotifers as a larval diet showed overall significantly lower growth rates. Growth was significantly enhanced by reduced salinity. Metabolic enzyme activity and relative myosin mRNA expression levels were not affected by larval diet. Muscle AAT and MDH were affected by salinity while these enzymes in liver tissue were affected by the interaction between salinity and temperature. Metabolic enzymes were stronger correlated with fish size than growth rates. Our results indicate that larval diet has a pronounced effect on juvenile growth rates under varying environmental conditions as optimal larval diet (zooplankton) increased juvenile growth rates significantly. Metabolic enzyme activity and relative myosin mRNA expression were not affected by larval history, which suggests that the persisting juvenile growth difference is not a result of differing metabolic capacity.

Protein synthesis is lowered while 20S proteasome activity is maintained following acclimation to low temperature in juvenile spotted wolffish (Anarhichas minor Olafsen).

The effects of temperature on protein metabolism have been studied mostly with respect to protein synthesis. Temperature generally has a parabolic effect on protein synthesis with a maximum rate being observed at optimal growth temperature. The effect of temperature on protein degradation is poorly understood. The 20S proteasome is mainly responsible for the degradation of short-lived and oxidatively modified proteins and has been recently identified as a potentially good proxy for protein degradation in fish. The aim of this experiment was to examine the relationships between the rate of protein synthesis, activity of the 20S proteasome, oxidative stress markers and antioxidant capacity in white muscle of juvenile spotted wolffish (Anarhichas minor) acclimated at three temperatures (4, 8 and 12 degrees C). The rate of protein synthesis was lower at 4 degrees C than at 8 degrees C while it was intermediate at 12 degrees C. Despite the decrease of protein synthesis at low temperature, the activity of 20S proteasome activity was maintained high in fish acclimated at lower temperature (4 degrees C), reaching levels 130% of that of fish acclimated at 8 degrees C when measured at a common temperature. The oxidative stress markers TBARS and protein-carbonyl content did not change among temperature groups, but reduced glutathione concentration was higher in cold-acclimated fish, suggesting a higher antioxidant capacity in this group. Our data suggest that lower growth rate in cold temperature results from both high 20S proteasome activity and a reduced rate of protein synthesis.

Ontogenesis of catabolic and energy metabolism capacities during the embryonic development of spotted wolffish (Anarhichas minor).

The catabolic and energy metabolism capacities during spotted wolffish (Anarhichas minor) embryogenesis were investigated. We assessed the embryo's ability to catabolize proteins (trypsin-like proteases) and lipids (triglyceride lipase) and examined the development of metabolic capacities using enzymatic assays: ability to use carbohydrates (pyruvate kinase), amino acids (aspartate aminotransferase) and fatty acids (hydroxyacyl-CoA dehydrogenase) for energy production, and aerobic (citrate synthase) and anaerobic (lactate dehydrogenase) energy production. Functional enzymatic systems were detected from the eyed stage (350 degree-days), except for fatty acids, which was detected from 540 degree-days. To compare the development of 1) aerobic and anaerobic pathways and 2) the capacity to mobilize the different energy substrates, enzymatic ratios were calculated. Anaerobic capacity appeared to increase at a significantly higher rate than the aerobic capacity. Ratios revealing the relative capacity to use specific energy substrates showed a significantly slower increase during development in the capacity to use carbohydrates than amino acids and fatty acids. The end of embryogenesis was characterized by a significant decrease in the use of carbohydrates for aerobic energy production but an increasing capacity to use amino acids. Egg survival as affected by the variability in metabolic parameters is discussed.

Early developmental plasticity and integrative responses in arctic charr (Salvelinus alpinus): effects of water velocity on body size and shape.

Environmental conditions such as temperature and water velocity may induce changes among alternative developmental pathways, i.e. phenotypic responses, in vertebrates. However, the extent to which the environment induces developmental plasticity and integrated developmental responses during early ontogeny of fishes remains poorly documented. We analyzed the responses of newly hatched Arctic charr (Salvelinus alpinus) to four experimental water velocities during 100 days of development. To our knowledge, this work is the first to analyze developmental plasticity responses of body morphology to an experimental gradient of water velocities during early ontogeny of fish. Arctic charr body size and shape responses show first, that morphometric traits display significant differences between low and high water velocities, thus revealing directional changes in body traits. Secondly, trait variation allows the recognition of critical ontogenetic periods that are most responsive to environmental constraints (40-70 and 80-90 days) and exhibit different levels of developmental plasticity. This is supported by the observation of asynchronous timing of variation peaks among treatments. Third, morphological interaction of traits is developmentally plastic and time-dependent. We suggest that developmental responses of traits plasticity and interaction at critical ontogenetic periods are congruent with specific environmental conditions to maintain the functional integrity of the organism.

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.).

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.