Acclimation to constant and fluctuating temperatures promotes distinct metabolic responses in Arctic char (Salvelinus alpinus).

The Arctic is warming three times faster than the global average, imposing challenges to cold-adapted fish, such as Arctic char (Salvelinus alpinus). We evaluated stress and metabolic responses of Arctic char to different thermal acclimation scenarios to determine whether responses to thermal variation differed from those to stable exposures. Fish were exposed for 7 days to one of four treatments: (1) control (12°C); (2) mean (16°C), corresponding to the mean temperature of the diel thermal cycle; (3) constant high temperature (20°C); and (4) diel thermal cycling (12 to 20°C every 24 h). Exposure to 20°C causes increases plasma lactate and glucose, an imbalance in antioxidant systems, and oxidative stress in the liver. The 20°C treatment also elevated fractional rates of protein synthesis and caused oxidative stress in the heart. Stress responses were more pronounced in diel thermal cycling than in mean (16°C) fish, indicating that peak exposure temperatures or variation are physiologically important. Cortisol was highest in diel thermal cycling fish and oxidative stress was noted in the liver. Gill Na+/K+-ATPase activity was also significantly reduced in diel thermal cycling fish, suggesting gill remodeling in response to an osmoregulatory stress. Exposure to a constant 20°C was more challenging than a diel thermal cycle, demonstrating the importance of daily cooling to recovery. Arctic char inhabit a thermally variable environment and understanding how this impacts their physiology will be critical for informing conservation strategies in the context of a rapidly warming Arctic.

The origins of gas exchange and ion regulation in fish gills: evidence from structure and function.

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid-base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills.

The role of salinity on genome-wide DNA methylation dynamics in European sea bass gills.

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change.

Salinity effects on expression and localization of aquaporin 3 in gills of the euryhaline milkfish (Chanos chanos).

Milkfish (Chanos chanos) are important euryhaline fish in Southeast Asian countries that can tolerate a wide range of salinity changes. Previous studies have revealed that milkfish have strong ion regulation and survival abilities under osmotic stress. In addition to ion regulation, water homeostasis in euryhaline teleosts is important during environmental salinity shifts. Aquaporins (AQP) are vital water channels in fish, and different AQPs can transport water influx or outflux from the body. AQP3 is one of the AQP channels, and the function of AQP3 in the gills of euryhaline milkfish is still unknown. The aim of this study was to investigate the expression and localization of AQP3 in the gills of euryhaline milkfish to contribute to our understanding of the physiological role and localization of AQP3 in fish. The AQP3 sequence was found in the milkfish next-generation sequencing (NGS) database and is mainly distributed in the gills of freshwater (FW)-acclimated milkfish. Under hypoosmotic and hyperosmotic stress, the osmolality of milkfish immediately shifted, similar to the aqp3 gene expression. Moreover, the abundance of AQP3 protein significantly decreased 3 h after transferring milkfish from FW to seawater (SW). However, there was no change within 7 days when the milkfish experienced hypoosmotic stress. Moreover, double immunofluorescence staining of milkfish gills showed that AQP3 colocalized with Na+ /K+ ATPase at the basolateral membrane of ionocytes. These results combined indicate that milkfish have a strong osmoregulation ability under acute osmotic stress because of the quick shift in the gene and protein expression of AQP3 in their gills.

Osmoregulatory Performance among Prickly Sculpin (Cottus asper) Living in Contrasting Osmotic Habitats.

AbstractDuring the colonization of freshwater by marine fish, adaptation to hypoosmotic conditions may impact their ability to osmoregulate in seawater. The prickly sculpin (Cottus asper) is a euryhaline fish with marine ancestors that postglacially colonized many freshwater habitats. Previous work on C. asper suggested that isolation in freshwater habitats has resulted in putative adaptations that improve ion regulation in freshwater populations compared with populations with current access to estuaries. To determine whether long-term colonization of freshwater is associated with a reduced ability to ion regulate in seawater, we acclimated C. asper populations from three habitat types that vary in the extent to which they are isolated from marine habitats and compared their seawater osmoregulation. Seawater acclimation revealed that lake populations exhibited a reduced capacity to osmoregulate in seawater compared with coastal river populations with ongoing access to estuaries. In particular, when acclimated to seawater for several weeks, lake populations had lower gill Na+/K+-ATPase activity and lower intestinal H+-ATPase activity than coastal river populations. Lake populations also had a reduced ability to maintain plasma ion concentrations, and they produced lower quantities of intestinal carbonate precipitates in seawater than coastal river populations. Furthermore, there was a positive relationship between the anterior intestinal Na+/K+-ATPase activity and the amount of precipitate produced by the intestine, which suggests that the anterior intestine plays a role in seawater osmoregulation. Our results suggest that the extent of isolation from the sea could, in part, explain the reduced osmoregulation in seawater in postglacial freshwater populations of C. asper.

Establishment and Characterization of a Novel Gill Cell Line, LG-1, from Atlantic Lumpfish (Cyclopterus lumpus L.).

The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Replacement of mitochondrion-rich cells during regeneration of the gills and opercular epithelium in zebrafish (Danio rerio).

Transport epithelia maintain the volume, ion concentration and acid-base balance of blood and extracellular fluids. In teleost fish, mitochondrion-rich cells (MRCs) are specialized ionocytes that perform this role. These cells are found in epithelia of the gills and buccal surface of the operculum (the bony structure covering the gills). Proliferation of MRCs in response to changes in water salinity and other environmental stressors is well documented, but the cellular mechanisms underlying MRC proliferation are poorly understood. Recently, regeneration and epithelial cell replacement in the gill filaments was demonstrated in the model vertebrate, zebrafish (Danio rerio), raising the question of whether MRCs are replaced during regrowth of transport epithelia. We chose two anatomical sites where MRCs are found-the gills and the opercular epithelium-to investigate whether MRCs were replaced following surgical resection of these structures. In live imaging experiments, we observed gradual replacement of the branchiostegal valve, an extension of the operculum, in zebrafish over a period of 21 days post-resection (dpr). In regenerating epithelia of both the operculum and gills, we detected MRCs by immunohistochemical localization of the α subunit of plasma membrane Na+/K+-ATPase. In both tissues, MRCs appeared soon after resection, and as early as 1 dpr in the gill filaments. We report regeneration of the operculum and proliferation of MRCs in regenerating tissue in adult zebrafish. These studies may contribute to our understanding of how MRC populations are regulated during the regenerative process, which may occur following exposure to environmental stressors, chemical toxicity or disease.

Acute and chronic temperature dependence of Na+/H+ exchange activity of Pimephales promelas gills.

Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes.

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.

Combined hypoxia and high temperature affect differentially the response of antioxidant enzymes, glutathione and hydrogen peroxide in the white shrimp Litopenaeus vannamei.

Low oxygen concentration in water (hypoxia) and high temperature are becoming more frequent due to climate change, forcing animals to endure stress or decease. Hypoxia and high temperature stress can lead to reactive oxygen species (ROS) accumulation and oxidative damage to the organisms. The shrimp Litopenaeus vannamei is the most cultivated crustacean worldwide. The aim of this study was to evaluate the expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT) and cytosolic manganese superoxide dismutase (cMnSOD) in gills and hepatopancreas from L. vannamei in response to two combined stressors: hypoxia and reoxygenation at control and high temperature (28 vs 35 °C, respectively). In addition, glutathione and hydrogen peroxide content were analyzed. The changes were mainly tissue-specific. In gills, cMnSOD expression and enzymatic activity increased in response to the interactions between oxygen variation and thermal stress, while GPx and CAT were maintained. More changes occurred in GPx, CAT and MnSOD in hepatopancreas than in gills, mainly due to the effect of the individual stress factors of thermal stress or oxygen variations. On the other hand, the redox state of glutathione indicated that during high temperature, changes in the GSH/GSSG ratio occurred due to the fluctuations of GSSG. Hydrogen peroxide concentration was not affected by thermal stress or oxygen variations in hepatopancreas, whereas in gills, it was not detected. Altogether, these results indicate a complex pattern of antioxidant response to hypoxia, reoxygenation, high temperature and their combinations.

Physiological performance of common carp (Cyprinus carpio, L., 1758) exposed to a sublethal copper/zinc/cadmium mixture.

In a natural ecosystem, fish are subjected to a multitude of variable environmental factors. It is important to analyze the impact of combined factors to obtain a realistic understanding of the mixed stress occurring in nature. In this study, the physiological performance of juvenile common carp (Cyprinus carpio) exposed for one week to an environmentally relevant metal mixture (4.8 µg/L of copper; 2.9 µg/L of cadmium and 206.8 µg/L of zinc) and to two temperatures (10 °C and 20 °C), were evaluated. After 1, 3 and 7 days, standard (SMR) and maximum metabolic rate (MMR) were measured and aerobic scope (AS) was calculated. In addition, hematocrit, muscle lactate, histology of the gills and metal accumulation in gills were measured. While SMR, MMR and AS were elevated at the higher temperature, the metal mixture did not have a strong effect on these parameters. At 20 °C, SMR transiently increased, but no significant changes were observed for MMR and AS. During metal exposure, hematocrit levels were elevated in the 20 °C group. The bioaccumulation of Cd in the gills reflected the increased metabolic rate at the higher temperature, with more accumulation at 20 °C than at 10 °C. Anaerobic metabolism was not increased, which corresponds with the lack of significant histopathological damage in the gill tissue. These results show that common carp handled these metal exposures well, although increased temperature led to higher Cd accumulation and necessitated increased hematocrit levels to maintain aerobic performance.

The potential for salt toxicity: Can the trans-epithelial potential (TEP) across the gills serve as a metric for major ion toxicity in fish?

An emerging Multi-Ion Toxicity (MIT) model for assessment of environmental salt pollution is based on the premise that major ion toxicity to aquatic organisms is related to a critical disturbance of the trans-epithelial potential across the gills (ΔTEP), which can be predicted by electrochemical theory. However, the model has never been evaluated physiologically. We directly tested key assumptions by examining the individual effects of eight different salts (NaCl, Na2SO4, MgCl2, MgSO4, KCl, K2SO4, CaCl2, and CaSO4) on measured TEP in three different fish species (fathead minnow, Pimephales promelas = FHM; channel catfish, Ictalurus punctatus = CC; bluegill, Lepomis macrochirus = BG). A geometric concentration series based on previously reported 96-h LC50 values for FHM was used. All salts caused concentration-dependent increases in TEP to less negative/more positive values in a pattern well-described by the Michaelis-Menten equation. The ΔTEP responses for different salts were similar to one another within each species when concentrations were expressed as a percentage of the FHM LC50. A plateau was reached at or before 100 % of the LC50 where the ΔTEP values were remarkably consistent, with only 1.4 to 2.2-fold variation. This relative uniformity in the ΔTEP responses contrasts with 28-fold variation in salt concentration (in mmol L-1), 9.6-fold in total dissolved solids, and 7.9-fold in conductivity at the LC50. The Michaelis-Menten Km values (salt concentrations causing 50 % of the ΔTEPmax) were positively related to the 96-h LC50 values. ΔTEP responses were not a direct effect of osmolarity in all species and were related to specific cation rather than specific anion concentrations in FHM. These responses were stable for up to 24 h in CC. The results provide strong physiological support for the assumptions of the MIT model, are coherent with electrochemical theory, and point to areas for future research.

The regulatory effects of pyridoxine deficiency on the grass carp (Ctenopharyngodon idella) gill barriers immunity, apoptosis, antioxidant, and tight junction challenged with Flavobacterium columnar.

The effects of dietary pyridoxine (PN) on the gill immunity, apoptosis, antioxidant and tight junction of grass cap (Ctenopharyngodon idella) were investigated in this study. Fish were fed semi-purified diets containing graded levels of PN for 10 weeks, and then challenged with Flavobacterium columnare by bath immersion exposure for 3 days. The results indicated that compared with the optimal PN level, PN deficiency resulted in a decline in the antimicrobial compound production of gill. In addition, PN deficiency up-regulated the pro-inflammatory cytokines and down-regulated the anti-inflammatory cytokines gene expression, which might be associated with the enhanced nuclear factor κB p65 and the inhibited target of rapamycin signalling pathways, respectively, suggesting that PN deficiency could impair gill immune barrier function. Furthermore, PN deficiency (1) induced cell apoptosis, which may be partly associated with the (apoptotic protease activating factor-1, Bcl-2 associated X protein)/caspase-9 and c-Rel/tumor necrosis factor α (rather than FasL)/caspase-8 mediated apoptosis pathway. (2) Inhibited Kelch-like ECH-associating protein 1a/NF-E2-related factor 2 mRNA expression, decreased the mRNA expression and activities of antioxidant enzymes, increased the levels of reactive oxygen species, protein carbonyl and malondialdehyde. (3) Increased the mRNA expression level of myosin light chain kinase, which may be result in the down-regulation of tight junction complexes such as zonula occludens 1, occludin and claudins (expect claudin-12 and claudin-15). These results suggest that PN deficiency could impair gill physical barrier function. In summary, dietary PN deficiency could cause the impairment of gill barrier function associated with immunity, apoptosis, antioxidant and tight junction, which may result in the increased the susceptibility of fish to pathogenic bacteria. Moreover, based on the gill rot morbidity, LZ activity and MDA content, the dietary PN requirements for grass cap were estimated to be 4.85, 4.78 and 4.77 mg kg-1 diet, respectively.

Hypoxia attenuate ionic transport in the isolated gill epithelium of Carcinus maenas.

The gills are osmorespiratory organs of aquatic organisms and the prime target of environmentally induced hypoxia. We have studied the impact of severe hypoxia (0.5 mg O2/L) on the ionic transport across posterior gills of Carcinus maenas acclimated to 12 ppt seawater (DSW). The short-circuit current (Isc) across hemilamellae from gills i.e. active ion transport was studied in micro Ussing chambers. Hypoxia induced by deoxygenation of the basolateral side, and not the apical side, resulted in time-dependent inhibition of Isc and full recovery of Isc after reoxygenation. Exposure of the crabs to severe 7 h hypoxia decreased the specific activity of Na+,K+-ATPase in the gills by 36%. Full recovery of enzyme activity occurred in fasted crabs after 3 days of reoxygenation. The intensity of Western blotting bands was not different in the gills of oxygenated, hypoxic and reoxygenated crabs. The reversible, nonspecific blocker of K+ channels Cs and hypoxia inhibited over 90% of Isc which is after reoxygenation fully recovered. The specific blocker of Cl- channels NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid] blocked Isc by 68.5%. Only the rest of not inhibited Isc in aerated saline was blocked by hypoxia and recovered after reoxygenation. The activity of the antioxidant enzyme catalase was not changed during hypoxia and reoxygenation kept the high enzyme activity in the gills at the level of crabs acclimated to DSW. As a response to hypoxia the presence of 2 mM H2O2 induce an initial slight transient decrease of Isc followed by a rise and after reoxygenation fully recovered Isc. Incubation of hemilamellae with the antioxidant derivative Trolox did not affect the inhibition of Isc by hypoxia.

Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass.

Acclimation to low salinities is a vital physiological challenge for euryhaline fish as the European sea bass Dicentrarchus labrax. This species undertakes seasonal migrations towards lagoons and estuaries where a wide range of salinity variations occur along the year. We have previously reported intraspecific differences in freshwater tolerance, with an average 30% mortality rate. In this study, we bring new evidence of mechanisms underlying freshwater tolerance in sea bass at gill and kidney levels. In fresh water (FW), intraspecific differences in mRNA expression levels of several ion transporters and prolactin receptors were measured. We showed that the branchial Cl-/HCO3- anion transporter (slc26a6c) was over-expressed in freshwater intolerant fish, probably as a compensatory response to low blood chloride levels and potential metabolic alkalosis. Moreover, prolactin receptor a (prlra) and Na+/Cl- cotransporter (ncc1) but not ncc-2a expression seemed to be slightly increased and highly variable between individuals in freshwater intolerant fish. In the posterior kidney, freshwater intolerant fish exhibited differential expression levels of slc26 anion transporters and Na+/K+/2Cl- cotransporter 1b (nkcc1b). Lower expression levels of prolactin receptors (prlra, prlrb) were measured in posterior kidney which probably contributes to the failure in ion reuptake at the kidney level. Freshwater intolerance seems to be a consequence of renal failure of ion reabsorption, which is not sufficiently compensated at the branchial level.

Impact of temperature shift on gill physiology during smoltification of Atlantic salmon smolts (Salmo salar L.).

Exposure to a temperature increase may disrupt smoltification and delay or stop the downstream migration of smolts. Thermal regimes are often different between a river and its tributaries, but the effects of a relative temperature shift are not well described. We used expression of smoltification genes coupled with gill Na+/K+-ATPase activity (NKA) and plasma cortisol and growth hormone (GH) levels to investigate the impact of a 5 °C difference between tributary and river on salmon juveniles. Responses to a temperature challenge were examined at four time points during the smoltification period, with juveniles reared under three regimes including control, early and late temperature increase. The temperature shifts reduced gill NKA, plasma GH and cortisol levels which indicate hypo-osmoregulation impairment and may reduce the survival of smolts. Out of the 22 genes examined, the expression of six genes was influenced by the temperature treatments, while changes in further eleven genes were influenced by the date of sampling. Genes usually known to be upregulated during smoltification were downregulated after the temperature increase, notably nkaα1b, nkcc1a and igf1r. Upregulation of some genes involved in the hormonal regulation and acid-base equilibrium in early June may indicate a switch towards desmoltification. This study gives further insights about the impact of temperature increase on the molecular processes underlying smoltification and possible responses to human-related water temperature increase. The data also suggest dual roles in the smoltification and desmoltification for GH and IGF1 and points to the implication of genes in the smoltification process, that have previously been unstudied (nbc) or with little data available (igf2).

The bimodal gas exchange strategies of dragonfly nymphs across development.

Dragonfly nymphs are aquatic and breathe water using a rectal gill. However, it has long been known that the nymphs of many species appear to possess the ability to breathe air, either during their final instar when they leave the water prior to metamorphosis, or during periods of aquatic hypoxia. The aerial gas exchange associated with these activities has not been quantified. This study used flow-through respirometry to measure the rate of aerial CO2 release (V̇CO2) from dragonfly nymphs as a proxy for their aerial gas exchange, both across development and in response to progressive aquatic hypoxia. It examined a total of four species from two families (Libellulidae and Aeshnidae). In both families, the late-final instar nymphs developed functional mesothoracic spiracles, allowing them to breathe air by positioning their head and thorax above the water's surface. While breathing air in this position, the nymphs could also ventilate their submerged rectal gill. Thus, during bimodal gas exchange in normoxic water, it was calculated that aeshnid nymphs expelled 39% of their respiratory CO2 into the air through their spiracles, while libellulid nymphs expelled 56% into the air. Decreasing the aquatic PO2 to 2.5 kPa and then below 1 kPa increased the proportion of respiratory CO2 expelled into the air from 69% to 100%, respectively. Thus, bimodally breathing late-final nymphs can vary how they partition gas exchange between their spiracles and their gill depending on aquatic PO2. Aeshnid nymphs of all developmental stages were also found to use their rectal gill as an air-breathing organ; pre-final nymphs performing 'surface skimming' while late final nymphs aspirated air bubbles directly into their gill's branchial basket. Mass-specific rates of aerial V̇CO2 also increased as the nymphs approached metamorphosis. These findings indicate that aeshnid nymphs are capable of accessing aerial O2 across development using their rectal gill as an air breathing organ, while the aquatic nymphs of both aeshnid and libellulid dragonflies undergo a progressive shift towards using the atmosphere for respiration as they approach metamorphosis.

Involvement of the phosphoryl transfer network in gill bioenergetic imbalance of pacamã (Lophiosilurus alexandri) subjected to hypoxia: notable participation of creatine kinase.

Hypoxia is among the most critical environmental stressors for fish in aquatic environments, and several energetic alterations have been associated with it. The aim of the present study was to evaluate the involvement of the phosphoryl transfer network and its effects on adenosine triphosphate (ATP)-dependent enzymes during hypoxia, as well as the role of oxidative stress in the activity of the phosphoryl transfer network in pacamã (Lophiosilurus alexandri) subjected to severe hypoxia. Branchial creatine kinase (CK; cytosolic and mitochondrial fractions), adenylate kinase (AK), and pyruvate kinase (PK) activities were inhibited after 72 h of exposure to hypoxia compared to their respective normoxia groups, and remained low (except for AK) after 24 and 72 h of re-oxygenation. Activities of the branchial sodium-potassium pump (Na+, K+-ATPase) and proton pump (H+-ATPase) were inhibited in fish exposed to 72 h of hypoxia compared to the normoxia group, remained inhibited after 24 h of re-oxygenation, and were restored to physiological levels after 72 h of re-oxygenation. Levels of branchial reactive oxygen species (ROS) were higher in fish exposed to hypoxia for 72 h compared to the normoxia group, and increased during re-oxygenation. Lipid peroxidation (LOOH) levels were higher in fish subjected to 72 h of hypoxia compared to the normoxia group, and remained higher during re-oxygenation. On the other hand, protein sulfhydryl (PSH) levels were lower in fish exposed to hypoxia for 72 h compared to the normoxia group, and remained low during re-oxygenation. Based on this evidence, inhibition of the activities of enzymes belonging to phosphoryl transfer network contributed to impairing energetic homeostasis linked to ATP production and ATP utilization in gills of pacamã subjected to hypoxia, and remained inhibited during re-oxygenation (except AK activity). Moreover, inhibition of the phosphoryl transfer network impaired activity of ATP-dependent enzymes, which can be mediated by ROS overproduction, lipid peroxidation, and oxidation of SH groups.

Changes in hypothermal stress-induced hepatic mitochondrial metabolic patterns between fresh water- and seawater-acclimated milkfish, Chanos chanos.

Milkfish (Chanos chanos) is a tropical euryhaline species. It can acclimate to fresh water (FW) or seawater (SW) and be cultured in both. In winter, cold snaps cause huge losses in milkfish revenue. Compared to FW-acclimated individuals, SW-acclimated milkfish have better low-temperature tolerance. Under hypothermal stress, a stable energy supply is critical to maintain normal liver function. In this study, the levels of key mitochondrial enzymes (citrate synthase (CS) and cytochrome c oxidase (COX)) in milkfish livers were examined. The CS:COX activity ratio in FW milkfish significantly increased under hypothermal stress (18 °C) whereas ATP (the end product of aerobic metabolism) was downregulated. Therefore, the activities of the enzymes involved in mitochondrial amino acid biosynthesis (aspartate aminotransferase (AST) and glutamate dehydrogenase (GDH)) were evaluated to elucidate energy flow in milkfish livers under hypothermal stress. In FW milkfish, GDH activity was upregulated whereas AST activity was downregulated. Nevertheless, the levels of all the aforementioned enzymes did not significantly change in SW milkfish under hypothermal stress. In summary, we clarified the mechanism accounting for the fact that SW milkfish have superior low-temperature tolerance to FW milkfish and demonstrated that SW and FW milkfish have different and unique strategies for regulating energy flow.

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation.

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro-CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix-rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.