Seawater Acclimation Research Articles

Sea water acclimation of rainbow trout (Oncorhynchus mykiss) modulates the mucosal transcript immune response induced by Vibrio anguillarum and Aeromonas salmonicida vaccine, and prevents further transcription of stress-immune genes in response to acute stress

Mucosal tissues appear to be more important in fish than in mammals due to living in a microbial-rich aquatic milieu, yet the complex interaction between the immune and the neuroendocrine system in these tissues remains elusive. The aim of this work was to investigate the mucosal immune response in immunized rainbow trout vaccinated with Alpha ject vaccine (bivalent), kept in fresh water (FW) or transferred to seawater (SW), and to evaluate their response to acute stress (chasing). Acute stress resulted in higher levels of plasma cortisol (Sham + Stress and Vaccine + Stress). A similar response was observed in skin mucus, but it was lower in Vaccine + Stress compared with stressed fish. With a few exceptions, minimal alterations were detected in the transcriptomic profile of stress-immune gene in the skin of vaccinated and stressed fish in both FW and SW. In the gills, the stress elicited activation of key stress-immune components (gr1, mr, β-ar, hsp70, c3, lysozyme, α-enolase, nadph oxidase, il1β, il6, tnfα, il10 and tgfβ1) in FW, but fewer immune changes were induced by the vaccine (nadph oxidase, il6, tnfα, il10 and igt) in both SW and FW. In the intestine, an array of immune genes was activated by the vaccine particularly those related with B cells (igm, igt) and T cells (cd8α) in FW with no stimulation observed in SW. Therefore, our survey on the transcriptomic mucosal response demonstrates that the immune protection conferred by the vaccine to the intestine is modulated in SW. Overall, our results showed: i) plasma and skin mucus cortisol showed no additional stress effect induced by prolonged SW acclimation, ii) the stress and immune response were different among mucosal tissues which indicates a tissue-specific response to specific antigens/stressor. Further, the results suggest that the systemic immune organs may be more implicated in infectious events in SW (as few changes were observed in the mucosal barriers of immunized fish in SW) than in FW.

Esophageal expression of Na+/H+ exchanger 2 and -3 (nhe2 and -3) gene transcripts in Atlantic salmon during smoltification and seawater acclimation

Atlantic salmon ( Salmo salar) undergo smoltification, a springtime transformation associated with the development of physiological, morphological, and behavioral traits that enable individuals to thrive in pelagic marine habitats. In particular, the capacity for salmon to maintain hydromineral balance in seawater is attained through the coordinated development of ion- and water-transporting processes in key organs such as the gill, kidney, and gastrointestinal tract. To combat dehydration, Atlantic salmon drink ambient seawater that is initially desalinated in the esophagus to enable subsequent solute-linked water absorption by the intestine. The molecular processes underlying esophageal desalination of imbibed seawater and their regulation during smoltification and following seawater exposure remain largely unresolved. The current study explored whether Na+/H+ exchangers in the esophagus may contribute to the removal of Na+ from luminal seawater. We first determined that both nhe2 and -3 were robustly expressed in the esophagus of smolts; nhe2 was also highly expressed in gill and stomach. Next, we sampled salmon undergoing smoltification and observed springtime elevations in branchial Na+/K+-ATPase activity, Na+/K+/2Cl- cotransporter 1 and cystic fibrosis transmembrane regulator 1 transcription that coincided with increased seawater tolerance. There were no parallel springtime changes in esophageal nhe2 or -3 expression; but nhe3 expression was enhanced in smolts after 2 days of seawater exposure. In a subsequent experiment, we observed elevated nhe2 and -3 expression in smolts that were long-term acclimated (2 weeks) to seawater versus freshwater. Collectively, our results indicate that Na+/H+ exchangers contribute to the desalination processes in the esophagus. The next challenge will be to determine whether essential “seawater-adapting hormones,” such as growth hormone and cortisol, regulate the expression of Na+/H+ exchangers in support of seawater acclimation. Supported by the National Science Foundation (IOS-1755131 to J.P.B.). National Science Foundation [IOS-1755131] This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

The GH/IGF axis in the sea lamprey during metamorphosis and seawater acclimation

How the growth hormone (GH)/insulin-like growth factor (IGF) system affects osmoregulation in basal vertebrates remains unknown. We examined changes in the expression of components of the GH/IGF axis and gill ion transporters during metamorphosis and following seawater (SW) exposure of sea lamprey. During metamorphosis, increases in gill nka and nkcc1 and salinity tolerance were accompanied by increases in pituitary gh, liver igf1, gill ghr and igf1, but not liver ghr. SW exposure of fully metamorphosed sea lamprey resulted in slight increases in plasma chloride concentrations after SW exposure, indicating a high level of SW tolerance, but no major changes in mRNA levels of gill ion transporters or components of the GH/IGF axis. Our results indicate that metamorphosis is a critical point in the lifecycle of sea lamprey for stimulation of the GH/IGF axis and is temporally associated with and likely promotes metamorphosis and SW tolerance.

The role of environmental salinity on Na+-dependent intestinal amino acid uptake in rainbow trout (Oncorhynchus mykiss)

Na+/K+-ATPases (NKA) in the basolateral membrane of the intestinal enterocytes create a Na+-gradient that drives both ion-coupled fluid uptake and nutrient transport. Being dependent on the same gradient as well as on the environmental salinity, these processes have the potential to affect each other. In salmonids, L-lysine absorption has been shown to be higher in freshwater (FW) than in seawater (SW) acclimated fish. Using electrophysiology (Ussing chamber technique), the aim was to explore if the decrease in L-lysine transport was due to allocation of the Na+-gradient towards ion-driven fluid uptake in SW, at the cost of amino acid transport. Intestinal NKA activity was higher in SW compared to FW fish. Exposure to ouabain, an inhibitor of NKA, decreased L-lysine transport. However, exposure to bumetanide and hydrochlorothiazide, inhibitors of Na+, K+, 2Cl−-co-transporter (NKCC) and Na+, Cl−-co-transporter (NCC) respectively, did not affect the rate of intestinal L-lysine transport. In conclusion, L-lysine transport is Na+-dependent in rainbow trout and the NKA activity and thus the available Na+-gradient increases after SW acclimation. This increased Na+-gradient is most likely directed towards osmoregulation, as amino acid transport is not compromised in SW acclimated fish.

Comparative Biology of Daphniopsis tibetana from Different Habitats under Seawater Acclimation

In this paper, we used experimental ecology methods and third-generation transcriptome sequencing to see the differences in growth, development, and reproduction of three strains of Daphniopsis tibetana Sars, 1903 from different locations in Tibet (Lake Namukacuo, NMKC; Lake Pengcuo, PC; and Lake Zigetangcuo, ZGTC). We also wanted to determine if the genes had changed after seawater-domesticated D. tibetana was reared in a laboratory. The results showed that at 15–16 ppt salinity and 15 ± 0.5 °C, the NMKC strain exhibited the highest survival rate of 26 d, and the ZGTC strain had the lowest survival rate at 53 days of culture. The body length was observed in NMKC (153.6 ± 12.1%), followed by PC (136.4 ± 16.1%), and then ZGTC (86.2 ± 7.6%). Combined, wild-type and seawater-acclimated D. tibetana produced 7252 DEGs, of which 4146 were up-regulated and 3106 were down-regulated. DEGs were subjected to gene ontology enrichment analysis. The DEGs were mainly enriched in single-organism localization, transporter activity, macromolecule localization, and organic substance transport. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis was also performed and revealed that the RNA transport, protein digestion and absorption, and protein processing in the endoplasmic reticulum pathways were highly enriched. The data mined can provide a reference for follow-up research.

Metabolic costs associated with seawater acclimation in a euryhaline teleost, the fourspine stickleback (Apeltes quadracus).

The cost of osmoregulation in teleosts has been debated for decades, with estimates ranging from one to 30 % of routine metabolic rate. The variation in the energy budget appears to be greater for euryhaline fish due to their ability to withstand dynamic salinity levels. In this study, a time course of metabolic and physiological responses of the euryhaline fourspine stickleback (Apeltes quadracus) acclimated to freshwater (FW) and then exposed to seawater (SW) was examined. There was 18% mortality in the first 3 days following exposure to SW, with no mortalities in the FW control group. Gill Na+/K+-ATPase (NKA) activity, an index of osmoregulatory capacity, increased 2.6-fold in SW fish peaking on days 7 and 14. Gill citrate synthase activity, an index of aerobic capacity, was 50-62% greater in SW than FW fish and peaked on day 7. Tissue water content was significantly lower in the SW fish on day 1 only, returning to FW levels by day 3. Routine metabolic rate was decreased within 24h of SW exposure and was maintained slightly (8-22%) but significantly lower in SW compared to FW water controls throughout the 2-week experiment. These results indicate that elevated salinity resulted in increased SW osmoregulatory and aerobic capacity in the gill, but with a reduced whole animal metabolic rate to this euryhaline species.

Juvenile sea lamprey (Petromyzon marinus) have a wide window of elevated salinity tolerance that is eventually limited during springtime warming

The present study examined changes in biometric characteristics, osmoregulatory capacity, and seawater (SW) tolerance of juvenile sea lamprey ( Petromyzon marinus) throughout the varying thermal changes from late autumn to late spring. Body length, mass, and condition factor were maintained until April, when significant declines in mass and condition factor were observed to correspond with increases in temperature. Nearly 100% survival in SW was maintained through April. In May, after river and estuarine temperatures had increased, significant mortality in SW (up to 50%) was observed. After SW acclimation, plasma chloride was maintained at an elevated set point, and gill Na+/K+-ATPase activity was elevated. Neither parameter appeared to be affected during springtime warming. Together, these results provide a first characterization of the sustained osmoregulatory performance of juvenile sea lamprey after metamorphosis and show that the window of increased hypo-osmoregulatory performance for SW entry lasts for at least 5 months but may ultimately be limited by increases in river water temperatures in late spring.

Na+/HCO3- cotransporter 1 (nbce1) isoform gene expression during smoltification and seawater acclimation of Atlantic salmon.

The life history of Atlantic salmon (Salmo salar) includes an initial freshwater phase (parr) that precedes a springtime migration to marine environments as smolts. The development of osmoregulatory systems that will ultimately support the survival of juveniles upon entry into marine habitats is a key aspect of smoltification. While the acquisition of seawater tolerance in all euryhaline species demands the concerted activity of specific ion pumps, transporters, and channels, the contributions of Na+/HCO3- cotransporter 1 (Nbce1) to salinity acclimation remain unresolved. Here, we investigated the branchial and intestinal expression of three Na+/HCO3- cotransporter 1 isoforms, denoted nbce1.1, -1.2a, and -1.2b. Given the proposed role of Nbce1 in supporting the absorption of environmental Na+ by ionocytes, we first hypothesized that expression of a branchial nbce1 transcript (nbce1.2a) would be attenuated in salmon undergoing smoltification and following seawater exposure. In two separate years, we observed spring increases in branchial Na+/K+-ATPase activity, Na+/K+/2Cl- cotransporter 1, and cystic fibrosis transmembrane regulator 1 expression characteristic of smoltification, whereas there were no attendant changes in nbce1.2a expression. Nonetheless, branchial nbce1.2a levels were reduced in parr and smolts within 2days of seawater exposure. In the intestine, gene transcript abundance for nbce1.1 increased from spring to summer in the anterior intestine, but not in the posterior intestine or pyloric caeca, and nbce1.1 and -1.2b expression in the intestine showed season-dependent transcriptional regulation by seawater exposure. Collectively, our data indicate that tissue-specific modulation of all three nbce1 isoforms underlies adaptive responses to seawater.

Low Omega-3 Levels in the Diet Disturbs Intestinal Barrier and Transporting Functions of Atlantic Salmon Freshwater and Seawater Smolts.

Due to a limited access to marine raw materials from capture fisheries, Atlantic salmon feeds are currently based on mainly plant ingredients (75%) while only 25% come from traditional marine ingredients including marine fish meal and fish oil. Thus, current feeds contain less of the essential omega-3 fatty acids. The aim of the study was to assess the impact of different omega-3 levels in fish feed on intestinal barrier and transporting functions of Atlantic salmon freshwater and seawater smolts. Atlantic salmon were fed three levels of omega-3 (2, 1 and 0.5%) and fish performance was followed through smoltification and the subsequent seawater acclimation. Intestinal barrier and transporting functions were assessed using Ussing chamber methodology and combined with transcript analysis of tight junction related proteins and ion transporters. A linear decrease in growth was observed with decreasing omega-3 levels. Low (0.5%) inclusion of omega-3 impaired the barrier function of the proximal intestine compared to 2% inclusion. Further, low levels of omega-3 decrease the transepithelial electrical potential across the epithelium indicating disturbed ion transport. It can be concluded that low dietary levels of omega-3 impair somatic growth and intestinal function of Atlantic salmon.

Neuroendocrine Regulation of Plasma Cortisol Levels During Smoltification and Seawater Acclimation of Atlantic Salmon

Diadromous fishes undergo dramatic changes in osmoregulatory capacity in preparation for migration between freshwater and seawater. One of the primary hormones involved in coordinating these changes is the glucocorticoid hormone, cortisol. In Atlantic salmon (Salmo salar), cortisol levels increase during the spring smoltification period prior to seawater migration; however, the neuroendocrine factors responsible for regulating the hypothalamic-pituitary-interrenal (HPI) axis and plasma cortisol levels during smoltification remain unclear. Therefore, we evaluated seasonal changes in circulating levels of cortisol and its primary secretagogue—adrenocorticotropic hormone (ACTH)—as well as transcript abundance of the major regulators of HPI axis activity in the preoptic area, hypothalamus, and pituitary between migratory smolts and pre-migratory parr. Smolts exhibited higher plasma cortisol levels compared to parr across all timepoints but circulating ACTH levels were only elevated in May. Transcript abundance of preoptic area corticotropin-releasing factor b1 and arginine vasotocin were ~2-fold higher in smolts compared to parr in February through May. Smolts also had ~7-fold greater hypothalamic transcript abundance of urotensin 1 (uts-1a) compared to parr in May through July. When transferred to seawater during peak smolting in May smolts rapidly upregulated hypothalamic uts-1a transcript levels within 24 h, while parr only transiently upregulated uts-1a 96 h post-transfer. In situ hybridization revealed that uts-1a is highly abundant in the lateral tuberal nucleus (NLT) of the hypothalamus, consistent with a role in regulating the HPI axis. Overall, our results highlight the complex, multifactorial regulation of cortisol and provide novel insight into the neuroendocrine mechanisms controlling osmoregulation in teleosts.

Tissue and salinity specific Na+/Cl\u2212 cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus)

Two orthologues of the gene encoding the Na+-Cl− cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl− cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

Effects of constant and diel cyclic temperatures on the liver and intestinal phospholipid fatty acid composition in rainbow trout Oncorhynchus mykiss during seawater acclimation

BackgroundRainbow trout is an economically important fish in aquaculture and is a model species in environmental physiology. Despite earlier research on the seawater adaptability of rainbow trout at different temperature regimes, the influence on the liver and intestine in this species is still unknown. Two trials were conducted to investigate the effects of constant and diel cyclic temperatures on phospholipid fatty acid (PLFA) composition in the liver and intestine of rainbow trout during seawater acclimation.ResultsAt the end of growth trial 1, fish at 9 and 12.5 °C showed significantly higher ratios of unsaturated to saturated (U/S) and unsaturation index (UI) than those at 16 °C in liver and intestine phospholipids. After day 1 of seawater acclimation, the U/S, UI, and average chain length (ACL) of liver and intestinal phospholipids in fish at 16 °C significantly increased. Two weeks after seawater acclimation, the liver and intestinal PLFA composition adapted to salinity changes. In trial 2, significantly higher U/S, UI, and ACL were found in intestinal phospholipids at 13 ± 2 °C. On the first day after seawater acclimation, UI and ACL in liver phospholipids significantly increased at 13 °C, while fish at 13 ± 2 °C showed significantly decreased U/S, UI, and ACL in the intestine. At the end of growth trial 2, liver PLFA compositions were stable, whereas intestinal PLFA at 13 and 13 ± 1 °C showed significantly decreased U/S, UI, and ACL. A two-way analysis of variance and principal component analysis revealed significant effects of different constant temperatures, seawater acclimation, and their interaction on the liver and intestinal phospholipids, a significant effect of diel cyclic temperature on intestinal phospholipids, and the effects of seawater acclimation and its interaction with diel cyclic temperature on liver phospholipids.ConclusionTemperatures of 9 and 12.5 °C could elevate membrane fluidity and thickness in the liver and intestine of rainbow trout in freshwater, whereas no significant effects were found with diel temperature variations. After seawater acclimation, constant and diel cyclic temperatures significantly influenced the membrane fluidity and thickness of the liver and intestine. Compared with constant temperature, diel temperature variation (13 ± 2 °C) can enhance the adaptability of rainbow trout during seawater acclimation.

Salinity-dependent expression of calcium-sensing receptors in Atlantic salmon (Salmo salar) tissues.

Multiple reports suggest that calcium-sensing receptors (CaSRs) are involved in calcium homeostasis, osmoregulation, and/or salinity sensing in fish (Loretz 2008, Herberger and Loretz 2013). We have isolated three unique full-length CaSR cDNAs from Atlantic salmon (Salmo salar) kidney that share many features with other reported CaSRs. Using anti-CaSR antibodies and PCR primers specific for individual salmon CaSR transcripts we show expression in osmoregulatory, neuroendocrine and sensory tissues. Furthermore, CaSRs are expressed in different patterns in salmon tissues where mRNA and protein expression are modified by freshwater or seawater acclimation. For example, in seawater, CaSR mRNA and protein expression is increased significantly in kidney as compared to freshwater. Electrophysiological recordings of olfactory responses produced upon exposure of salmon olfactory epithelium to CaSR agonists suggest a role for CaSRs in chemoreception in this species consistent with other freshwater, anadromous, and marine species where similar olfactory responses to divalent and polyvalent cations have been reported. These data provide further support for a role of CaSR proteins in osmoregulatory and sensory functions in Atlantic salmon, an anadromous species that experiences a broad range of environmental salinities in its life history.

The effects of temperature and salinity on the endocrinology in two life stages of juvenile rainbow/steelhead trout (Oncorhynchus mykiss)

The San Francisco Bay Delta is experiencing seasonally warmer waters and saltwater intrusion into historically freshwater ecosystems due to climate change. Steelhead/rainbow trout (Oncorhynchus mykiss) are resident in the Bay-Delta from juvenile development through the smoltification process. Due to increases in sea level, premature seawater (SW) acclimation may co-occur with increased temperatures on pre-smolt juveniles. To evaluate the interactive effects of salinity and temperature on juvenile life stages of salmonids, rainbow trout alevin (3 days post-hatching) were exposed to 13, 16.4 and 19°C for 10 days and then challenged for 24 h to 18 parts per thousand SW. Similarly, fry (4 weeks post-hatching) were exposed to 13, 16.4 and 19°C for 2 weeks (14 days) and then challenged to SW. Estradiol-17β (E2 ), cortisol, triiodothyronine (T3 ) and thyroxine (T4 ) were measured in whole animal homogenates and muscle tissue using enzyme-linked immunosorbent assays. Transcripts of gill Na+ /K+ ATPase β (NKAα1b), brain growth hormone I (gh1) and brain gonadotropin-releasing hormone receptor 2 (gnrh2) were also measured. Alevin exhibited a significant temperature-dependent decrease in survival, and fry showed a temperature-dependent decrease in condition factor. The gene expression of NKAα1b, gh1 and gnrh2 significantly decreased in all SW-challenged alevin, and a significant decrease in gnrh2 expression was observed in fry with temperature. Alevin T3 and T4 concentrations significantly increased with increasing temperature. There was a temperature-dependent increase in E2 of fry but not of alevin. The results of this study demonstrate that increasing temperature and SW exposure may adversely affect the survival and SW acclimation of alevin and fry stages of salmonids and that the tolerances of younger juvenile stages should be considered when assessing the response of salmonid populations to climate change stressors.

Effects of different temperatures on seawater acclimation in rainbow trout Oncorhynchus mykiss: osmoregulation and branchial phospholipid fatty acid composition.

This study aimed to investigate the effects of different temperatures on seawater acclimation in rainbow trout (Oncorhynchus mykiss), in terms of growth performance, osmoregulatory capacity, and branchial phospholipid fatty acid(PLFA) composition. The fish (initial weight, 94.73g) were reared at 9, 12.5, and 16°C for 28days, then acclimated to seawater, and subsequently reared for 14days. Sampling points were on the last day in freshwater, and the 1st, 4th, 7th, and 14th days after the salinity reached 30. The results showed the final weight, percent weight gain, and specific growth rate of rainbow trout at 12.5°C were significantly higher than those at 9°C, while the thermal growth coefficient at 16°C was significantly lower than that in other treatments. The branchial PLFA composition in rainbow trout changed more rapidly at 9 and 12.5°C than at 16°C. The branchial PLFA composition was significantly affected by temperature and salinity and their interaction. The polyunsaturated fatty acid content of phospholipids in the gill at 9 and 12.5°C was significantly higher than those at 16°C. Low temperature (9°C) and seawater acclimation significantly increased the degree of unsaturation of membrane, enhancing membrane fluidity, which is related to Na+-K+ ATPase activity. Responses of plasma ion, Na+-K+ ATPase activity, and plasma glucose followed a similar pattern at different temperatures. Overall, the study suggests that 12.5°C is the ideal temperature for seawater acclimation in rainbow trout.

Effects of seawater acclimation at constant and diel cyclic temperatures on growth, osmoregulation and branchial phospholipid fatty acid composition in rainbow trout Oncorhynchus mykiss.

The study investigated the effects of seawater acclimation at constant and diel temperatures on the growth, osmoregulation, and branchial phospholipid fatty acid (PLFA) composition in rainbow trout (Oncorhynchus mykiss). The fish (initial weight, 62.28 ± 0.41g) were reared at a constant 13.0°C (CT) or with a diel cycle of either 13.0 ± 1.0°C (VT2) or 13.0 ± 2.0°C (VT4) for 6weeks, and subsequently subjected to seawater acclimation. Diel temperature variations (of up to 4°C) did not affect the growth rate of rainbow trout maintained in freshwater, but alleviated the impairment on the growth after seawater challenge. Under all temperature conditions, rainbow trout were well prepared to seawater acclimation. The diel cyclic temperature resulted in fish with reduced fluctuations in plasma electrolyte levels, branchial Na+-K+ ATPase activity, and plasma osmolality. In freshwater, the sum of the monounsaturated fatty acids was significantly higher in the VT4 relative to CT and VT2 treatment. Conversely, the sum of polyunsaturated fatty acids was significantly lower in the VT4 fish. After seawater transfer, the branchial PLFA profiles of the fish significantly changed, but those in CT and VT2 did not recover afterwards (the degree of unsaturation was downregulated). The PLFA composition of fish in the VT4 treatment appeared to be steadier under seawater acclimation. This study suggests that a diel cyclic temperature (13.0 ± 2.0°C) can alleviate the impairment of growth, enhance osmoregulation capability, and improve the stability of the branchial PLFA composition in rainbow trout after seawater acclimation.

Effects of seawater acclimation on two Na+/K+-ATPase α-subunit isoforms in the gills of the marble goby, Oxyeleotris marmorata

The marble goby, Oxyeleotris marmorata, is a freshwater teleost, but can acclimate progressively to survive in seawater (salinity 30). As an obligatory air-breather, it can also survive long periods of emersion. Two isoforms of Na+/K+-ATPase (nka) α-subunit, nkaα1 and nkaα3, but not nkaα2, had been cloned from the gills of O. marmorata. The cDNA sequence of nkaα1 consisted of 3069 nucleotides, coding for 1023 amino acids (112.5 kDa), whereas nkaα3 consisted of 2976 nucleotides, coding for 992 amino acids (109.5 kDa). As only one form of branchial Nkaα1 was identified using molecular cloning in this study, O. marmorata lacks specific freshwater- and seawater-type Nkaα isoforms as demonstrated by some other euryhaline fish species. The nkaα1 transcript level was about 2.5-fold higher than that of nkaα3 in the gills of freshwater O. marmorata. During exposure to seawater, the branchial transcript level of nkaα1 increased significantly on day 1 (~3.3-fold) and day 6 (~2.6-fold). By contrast, the branchial transcript level of nkaα3 increased significantly on day 1 (~2.6-fold), but not on day 6, of seawater exposure. Six days of exposure to seawater also led to significant increases in protein abundances of Nkaα1 (~6.9-fold) and Nkaα3 (~2.8-fold) in the gills of O. marmorata. Hence, the mRNA and protein expressions of both nkaα1/Nkaα1 and nkaα3/Nkaα3 were up-regulated in O. marmorata during seawater acclimation. This could explain why Vmax increases but Km for Na+ and K+ remain unchanged in Nka extracted from the gills of O. marmorata acclimated to seawater as reported previously.

Capacity for freshwater acclimation and differences in the transcription of ion transporter genes underlying different migratory life histories of Takifugu fish.

The genus Takifugu is a group of approximately 20 species of puffer fishes living in a wide range of salinity environments around East Asian countries. This group presents a broad spectrum of evolutionary stages adapted to anadromy as a result of speciation that occurred a short time (2-5 million years) ago on an evolutionary timescale. This group thus can be considered as a model for studying the evolutionary mechanisms of anadromy. We firstly conducted a transfer experiment from seawater to low-salinity waters on five Takifugu species: two anadromous species T. obscurus and T. ocellatus, two euryhaline wanderer marine species T. rubripes and T. niphobles, and a strictly marine species T. snyderi, and confirmed that the capacity for acclimation to hypotonic environments was associated with their life history strategies. Next, transcriptomes of the gill and intestine of these species in hypotonic condition were compared to those under hypertonic condition for each species using RNA-Sequencing so as to determine possible candidate transporters playing an important role on freshwater adaptation. As this analysis suggested that cftr, encoding an important ion transporter for seawater acclimation in the gill, and ncc, encoding a transporter that is suggested to play important osmoregulatory roles in the intestine, are important candidates, their expression was validated by quantitative real-time PCR analysis. Expression of cftr was downregulated in the gills of the four euryhaline species under the hypotonic condition, but no change was detected in the gill of stenohaline T. snyderi, which may be one reason for the poor hypotonic acclimation capacity of T. snyderi. Expression of ncc was clearly upregulated in the intestines of the two anadromous species under the hypotonic condition, but not in other three species. Different ion transporter expression patterns between the five species indicate that the transcriptional regulation of cftr in the gill and ncc in the intestine may be important for the improvement of hypotonic acclimation capacity and evolution of anadromy in the Takifugu species.

Intestinal Gene Expression of Na+/HCO3− cotransporter 1 (nbce1) Isoforms in Atlantic Salmon During Smoltification and Seawater Acclimation

In preparation for migration from freshwater to marine habitats, Atlantic salmon undergo smoltification, a springtime transformation that includes the development of seawater tolerance. The ability of smolts to inhabit seawater is attained through the coordinated development of ion and water‐transporting processes in key organs such as the gill, kidney, and intestine. To promote solute‐linked water absorption, the intestinal epithelium of fishes acclimated to SW secretes HCO3− to form luminal Ca2+ and Mg2+ precipitates. As a source of HCO3− for apical anion exchange, Na+/HCO3− cotransporter 1 (Nbce1) facilitates the transport of plasma HCO3− into the interior of epithelial cells across the basolateral membrane. While Atlantic salmon (Salmo salar) express three nbce1 isoforms (nbce1.1, nbce1.2a, and nbce1.2b), there is no information on whether their expression corresponds with the development of intestinal fluid absorption that occurs during smoltification. We first determined that nbce1.1 and nbce1.2b gene transcripts were highly expressed in pyloric cacae and anterior intestine of smolts, whereas nbce1.2a showed low expression in the intestine but robust expression in gill. Next, we sampled salmon undergoing parr‐smolt transformation and observed elevated branchial Na+/K+‐ATPase activity in May characteristic of smoltification. Moreover, smolts exhibited an improved ability to maintain ionoregulatory balance when transferred to seawater in May versus March. We will report corresponding patterns of nbce1.1 and nbce1.2b expression in pyloric cacae and anterior intestine during smoltification and following seawater transfer to resolve whether their transcriptional patterns correspond with the acquisition of intestinal fluid absorptive processes.Support or Funding InformationSupported by the National Science Foundation (IOS‐1755131).