Euryhaline Teleost Research Articles

Transcriptional regulation of prolactin in a euryhaline teleost: Characterisation of gene promoters through in silico and transcriptome analyses.

The sensitivity of prolactin (Prl) cells of the Mozambique tilapia (Oreochromis mossambicus) pituitary to variations in extracellular osmolality enables investigations into how osmoreception underlies patterns of hormone secretion. Through the actions of their main secretory products, Prl cells play a key role in supporting hydromineral balance of fishes by controlling the major osmoregulatory organs (ie, gill, intestine and kidney). The release of Prl from isolated cells of the rostral pars distalis (RPD) occurs in direct response to physiologically relevant reductions in extracellular osmolality. Although the particular signal transduction pathways that link osmotic conditions to Prl secretion have been identified, the processes that underlie hyposmotic induction of prl gene expression remain unknown. In this short review, we describe two distinct tilapia gene loci that encode Prl177 and Prl188 . From our in silico analyses of prl177 and prl188 promoter regions (approximately 1000bp) and a transcriptome analysis of RPDs from fresh water (FW)- and seawater (SW)-acclimated tilapia, we propose a working model for how multiple transcription factors link osmoreceptive processes with adaptive patterns of prl177 and prl188 gene expression. We confirmed via RNA-sequencing and a quantitative polymerase chain reaction that multiple transcription factors emerging as predicted regulators of prl gene expression are expressed in the RPD of tilapia. In particular, gene transcripts encoding pou1f1, stat3, creb3l1, pbxip1a and stat1a were highly expressed; creb3l1, pbxip1a and stat1a were elevated in fish acclimated to SW vs FW. Combined, our in silico and transcriptome analyses set a path for resolving how adaptive patterns of Prl secretion are achieved via the integration of osmoreceptive processes with the control of prl gene transcription.

Gene expression profiling of proximal and distal renal tubules in Atlantic salmon (Salmo salar) acclimated to fresh water and seawater.

Euryhaline teleost kidneys undergo a major functional switch from being filtratory in freshwater (FW) to being predominantly secretory in seawater (SW) conditions. The transition involves both vascular and tubular effects. There is consensus that the glomerular filtration rate is greatly reduced upon exposure to hyperosmotic conditions. Yet, regulation at the tubular level has only been examined sporadically in a few different species. This study aimed to obtain a broader understanding of transcriptional regulation in proximal versus distal tubular segments during osmotic transitions. Proximal and distal tubule cells were dissected separately by laser capture microdissection, RNA was extracted, and relative mRNA expression levels of >30 targets involved in solute and water transport were quantified by quantitative PCR in relation to segment type in fish acclimated to FW or SW. The gene categories were aquaporins, solute transporters, fxyd proteins, and tight junction proteins. aqp8bb1, aqp10b1, nhe3, sglt1, slc41a1, cnnm3, fxyd12a, cldn3b, cldn10b, cldn15a, and cldn12 were expressed at a higher level in proximal compared with distal tubules. aqp1aa, aqp1ab, nka-a1a, nka-a1b, nkcc1a, nkcc2, ncc, clc-k, slc26a6C, sglt2, fxyd2, cldn3a, and occln were expressed at a higher level in distal compared with proximal tubules. Expression of aqp1aa, aqp3a1, aqp10b1, ncc, nhe3, cftr, sglt1, slc41a1, fxyd12a, cldn3a, cldn3b, cldn3c, cldn10b, cldn10e, cldn28a, and cldn30c was higher in SW- than in FW-acclimated salmon, whereas the opposite was the case for aqp1ab, slc26a6C, and fxyd2. The data show distinct segmental distribution of transport genes and a significant regulation of tubular transcripts when kidney function is modulated during salinity transitions.

Transcriptomic Analysis of Gill and Kidney from Asian Seabass (Lates calcarifer) Acclimated to Different Salinities Reveals Pathways Involved with Euryhalinity

Asian seabass (or commonly known as barramundi), Lates calcarifer, is a bony euryhaline teleost from the Family Latidae, inhabiting nearshore, estuarine, and marine connected freshwaters throughout the tropical Indo-West Pacific region. The species is catadromous, whereby adults spawn in salinities between 28 and 34 ppt at the mouth of estuaries, with resultant juveniles usually moving into brackish and freshwater systems to mature, before returning to the sea to spawn again as adults. The species lives in both marine and freshwater habitats and can move quickly between the two; thus, the species’ ability to tolerate changes in salinity makes it a good candidate for studying the salinity acclimation response in teleosts. In this study, the transcriptome of two major osmoregulatory organs (gills and kidneys) of young juvenile Asian seabass reared in freshwater and seawater were compared. The euryhaline nature of Asian seabass was found to be highly pliable and the moldability of the trait was further confirmed by histological analyses of gills and kidneys. Differences in major expression pathways were observed, with differentially expressed genes including those related to osmoregulation, tissue/organ morphogenesis, and cell volume regulation as central to the osmo-adaptive response. Additionally, genes coding for mucins were upregulated specifically under saline conditions, whereas several genes important for growth and development, as well as circadian entrainment were specifically enriched in fish reared in freshwater. Routing of the circadian rhythm mediated by salinity changes could be the initial step in salinity acclimation and possibly migration in euryhaline fish species such as the Asian seabass.

Potential osmoprotective roles of branchial heat shock proteins towards Na+, K+-ATPase in milkfish (Chanos chanos) exposed to hypotonic stress

In euryhaline teleosts, osmoregulatory mechanisms vary with osmotic stresses, and heat shock proteins (HSPs) play a central role in maintaining cellular homeostasis. The present study aimed to investigate the expression and potential roles of HSP70 and HSP90 in the gills of seawater (SW)- and freshwater (FW)-acclimated milkfish (Chanos chanos). Four HSP genes, including Cchsc70 (heat shock cognate 70), Cchsp70, Cchsp90α, and Cchsp90β, were analyzed in milkfish gills. Among these genes, only the mRNA abundance of branchial Cchsp90α was significantly lower in the FW-acclimated than in the SW-acclimated milkfish. Immunoblotting showed no significant difference in the relative protein abundance of branchial HSP70 and HSP90 between the two groups. The time-course experiments (from SW to FW) showed that the protein abundance of HSP70 and HSP90 at the 3 h and 6 h post-transfer and then declined gradually. To further illustrate the potential osmoregulatory roles of HSP70 and HSP90, their interaction with Na+, K+-ATPase (NKA, the primary driving force for osmoregulation) was analyzed using co-immunoprecipitation. The results showed the interaction between HSP70, HSP90 and NKA after acclimation to SW or FW increased within 3 h; and then returned to normal levels within 7 days. To our knowledge, the present study was the first to demonstrate that the interaction between HSP70, HSP90 and NKA changes with hypotonic stress in euryhaline teleosts. Before the transfer, no interaction was detected. When transferred to FW from SW, the interaction of HSP70 and HSP90 with NKA were detected. The results suggested that HSP70 and HSP90 participated in the acute responses of osmoregulatory mechanisms to protect branchial NKA from hypotonic stress in milkfish.

Differential effects of hypothermal stress on lactate metabolism in fresh water- and seawater-acclimated milkfish, Chanos chanos

The milkfish Chanos chanos, an economically important cultured marine species in Southeast Asia, exhibits stenothermal and euryhaline characteristics and huge mortality usually occurs during extreme cold weather in winter. Under conditions beyond optimal temperatures, ectothermic species experience an increase in anaerobic glycolysis. To better understand the hypothermal acclimation response of this tropical species, the lactate metabolic profiles of freshwater (FW)- and seawater (SW)-acclimated milkfish were compared under control (optimal temperature; 28 °C) and hypothermal treatment (18 °C) conditions. In this study, the lactate dehydrogenase (LDH) isoform genes, ldha and ldhb, were identified in milkfish livers and muscles, respectively. The LDH is a bidirectional enzyme that triggered the conversion of pyruvate to lactate via anaerobic glycolysis as LDH exhibits the reductase activity (LDH-R), while via the reverse direction as LDH exhibits the oxidase activity (LDH-O). The hypothermal stress significantly upregulated the LDH-R activity in the muscles and the monocarboxylate transporter activity in both muscles and livers, of SW- and FW-acclimated milkfish. The levels of blood lactate, however, decreased in SW-acclimated milkfish. Under hypothermal stress, anaerobic metabolism increased in the muscles of both FW and SW individuals, whereas the liver of SW-acclimated milkfish showed better acute phase capacity to utilize blood lactate than FW-acclimated milkfish. Taken together, in the present study, the major functions of the bidirectional enzyme LDH were identified according to its LDH-O and LDH-R activities. Furthermore, environmental salinities were found to affect the acute anaerobic metabolic strategies of euryhaline teleosts under hypothermal stress and were correlated with their hypothermal tolerance ability.

Energy and nitrogenous waste from glutamate/glutamine catabolism facilitates acute osmotic adjustment in non-neuroectodermal branchial cells

Maintenance of homeostasis is one of the most important physiological responses for animals upon osmotic perturbations. Ionocytes of branchial epithelia are the major cell types responsible for active ion transport, which is mediated by energy-consuming ion pumps (e.g., Na+-K+-ATPase, NKA) and secondary active transporters. Consequently, in addition to osmolyte adjustments, sufficient and immediate energy replenishment is essenttableial for acclimation to osmotic changes. In this study, we propose that glutamate/glutamine catabolism and trans-epithelial transport of nitrogenous waste may aid euryhaline teleosts Japanese medaka (Oryzias latipes) during acclimation to osmotic changes. Glutamate family amino acid contents in gills were increased by hyperosmotic challenge along an acclimation period of 72 hours. This change in amino acids was accompanied by a stimulation of putative glutamate/glutamine transporters (Eaats, Sat) and synthesis enzymes (Gls, Glul) that participate in regulating glutamate/glutamine cycling in branchial epithelia during acclimation to hyperosmotic conditions. In situ hybridization of glutaminase and glutamine synthetase in combination with immunocytochemistry demonstrate a partial colocalization of olgls1a and olgls2 but not olglul with Na+/K+-ATPase-rich ionocytes. Also for the glutamate and glutamine transporters colocalization with ionocytes was found for oleaat1, oleaat3, and olslc38a4, but not oleaat2. Morpholino knock-down of Sat decreased Na+ flux from the larval epithelium, demonstrating the importance of glutamate/glutamine transport in osmotic regulation. In addition to its role as an energy substrate, glutamate deamination produces NH4+, which may contribute to osmolyte production; genes encoding components of the urea production cycle, including carbamoyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), were upregulated under hyperosmotic challenges. Based on these findings the present work demonstrates that the glutamate/glutamine cycle and subsequent transepithelial transport of nitrogenous waste in branchial epithelia represents an essential component for the maintenance of ionic homeostasis under a hyperosmotic challenge.

Adaptive evolution of low-salinity tolerance and hypoosmotic regulation in a euryhaline teleost, Takifugu obscurus

The mechanism of osmoregulation is crucial for maintaining growth, development, and life activities in teleosts. Takifugu obscurus, the only euryhaline species in the genus Takifugu, is a proper model organism for studying the mechanism of low-salt tolerance and hypoosmotic regulation. In this study, whole-genome sequencing data were obtained from 90 pufferfish representing five species within this genus, T. rubripes, T. obscurus, T. flavidus, T. niphobles, and T. bimaculatus. Using a phylogeny, PCA, and population structure analyses, we observed similar amounts of population genetic differentiation among species. The five species are closely related to each other and have differentiated within a relatively short period, while T. bimaculatus and T. flavidus shared the most similar genetic backgrounds. We further identified hundreds of genes under selection related to hypoosmotic regulation between T. obscurus and other Takifugu species, including 16 representative genes involving ion transporters (atp1a3, atp2a2, atp2a3, slc13a1, slc5a8, slc12a2, slc12a4, slc26a2, scn1b, and kcna2/3/10), genes involved in hormone regulation (fyn, prlr, and grb2), and a gene associated with water absorption (aqp3). Our findings provide preliminary insight into the mechanism of osmoregulation and will facilitate follow-up validation of candidate genes related to osmoregulation in T. obscurus.

A review on the activity of Na+/K+-ATPase in branchial ionocytes and its role in salinity adaptation among diadromous species

Freshwater and saltwater environments have opposite osmotic gradients. In freshwater, organisms tend to uptake ions from the surrounding, while in saltwater, they need to excrete the excess ions. Branchial ionocytes play a significant role in euryhaline teleost species that migrate to different salinity gradients as part of their life cycle. Ionocytes are highly dependent on the activity of Na+/K+-ATPase (NKA) in salinity adaptation. Thus, this review article determines the factors affecting NKA activity in ionocytes and its role in salinity adaptation among diadromous species. Several findings suggest that NKA activity is affected by various factors such as hormones, nutrition, and gene expression. Ionocytes enriched with NKA have many functions in diadromous depending on their types, location, size, and number in branchial necessary for these organisms to carry out their biological function in ionic-regulatory processes.

Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl− secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl− secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl− secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (Isc, equal to Cl− secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.

Characterization of Full-Length Transcriptome Sequences and Splice Variants of Lateolabrax maculatus by Single-Molecule Long-Read Sequencing and Their Involvement in Salinity Regulation.

Transcriptome complexity plays crucial roles in regulating the biological functions of eukaryotes. Except for functional genes, alternative splicing and fusion transcripts produce a vast expansion of transcriptome diversity. In this study, we applied PacBio single-molecule long-read sequencing technology to unveil the whole transcriptome landscape of Lateolabrax maculatus. We obtained 28,809 high-quality non-redundant transcripts, including 18,280 novel isoforms covering 8,961 annotated gene loci within the current reference genome and 3,172 novel isoforms. A total of 10,249 AS events were detected, and intron retention was the predominant AS event. In addition, 1,359 alternative polyadenylation events, 3,112 lncRNAs, 29,609 SSRs, 365 fusion transcripts, and 1,194 transcription factors were identified in this study. Furthermore, we performed RNA-Seq analysis combined with Iso-Seq results to investigate salinity regulation mechanism at the transcripts level. A total of 518 transcripts were differentially expressed, which were further divided into 8 functional groups. Notably, transcripts from the same genes exhibited similar or opposite expression patterns. Our study provides a comprehensive view of the transcriptome complexity in L. maculatus, which significantly improves current gene models. Moreover, the diversity of the expression patterns of transcripts may enhance the understanding of salinity regulatory mechanism in L. maculatus and other euryhaline teleosts.

Spotted Sea Bass (Lateolabrax maculatus) cftr, nkcc1a, nkcc1b and nkcc2: Genome-Wide Identification, Characterization and Expression Analysis Under Salinity Stress

The Na+/K+/2Cl− cotransporter (NKCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) proteins play crucial roles in the transportation of Na+ and Cl−. In this study, we identified cftr, nkcc1a, nkcc1b and nkcc2 in spotted sea bass (Lateolabrax maculatus) genomic and transcriptomic databases. We also characterized these genes via phylogenetic and structural analyses. The results showed that both cftr and nkcc were highly conservative in L. maculatus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis in ten tissues showed that cftr, nkcc1a and nkcc2 highly express in osmoregulatory organs such as gill, kidney and intestine. Furthermore, the expressions of cftr and nkcc1a in gill as well as nkcc2 in intestine were up-regulated by high salinity, indicating that these genes function potentially in osmoregulation. Our findings provided the insights into the cftr and nkcc functions in euryhaline teleost.

Acute ammonia toxicity and the interactive effects of ammonia and salinity on the standard metabolism of European sea bass (Dicentrarchus labrax)

Ammonia tolerance of European sea bass (Dicentrarchus labrax) juveniles (1.1 ± 0.3 g) was investigated by a series of acute toxicity experiments at different salinity levels (10, 20 and 30 ppt) at pH 8.0 and temperature 22 °C. Oxygen consumption rates were also measured to determine the Standard Metabolism (SM) of the fish. The tolerance of the fish to total ammonia nitrogen (TAN) and unionized ammonia (NH3) increased significantly with increasing salinity levels. The safe level for European sea bass was estimated to be 0.4, 1.0 and 1.6 mg L−1 for TAN and 0.02, 0.04 and 0.07 mg L−1 for NH3 at 10, 20 and 30 ppt salinity levels, respectively. At any, each level, the SM of the fish increased with increasing TAN concentration (P < .01). The SM of the fish at 10, 20 and 30 ppt gradients is 284, 282 and 272 mg h−1 kg−1, respectively at 0 mg L−1 TAN concentration (P < .01). This study indicates that European sea bass, a euryhaline teleost fish, has a low tolerance to ammonia at any salinity level.

Functionalized silver nanoparticles depress aerobic metabolism in the absence of overt toxicity in brackish water killifish, Fundulus heteroclitus

Engineered nanomaterials (ENMs) tend to precipitate in saline waters so the majority of aquatic toxicity studies have focused on freshwaters, where bioavailability is presumed to be higher. Recent studies have illustrated that some ENM formulations are bioavailable and bioactive in salt water and that their effects are more pronounced at the physiological than biochemical level. These findings raise concerns regarding the effects of ENMs on marine organisms. Therefore, our goal was to characterize the effects of polyvinylpyrolidone-functionalized silver ENMs (nAg) on aerobic performance in the killifish (Fundulus heteroclitus), a common euryhaline teleost. Fish were exposed to 80 μg L−1 of 5 nm nAg for 48 h in brackish water (12 ppt) and routine (ṀO2min) and maximum (ṀO2max) rates of oxygen consumption were quantified. Silver dissolution was minimal and nAg remained well dispersed in brackish water, with a hydrodynamic diameter of 21.0 nm, compared to 19.3 in freshwater. Both ṀO2min and ṀO2max were significantly lower (by 53 and 30%, respectively) in killifish exposed to nAg and a reduction in ṀO2 variability suggested spontaneous activity was suppressed. Neither gill Na+/K+-ATPase activity, nor various other biochemical markers were affected by nAg exposure. The results illustrate that a common ENM formulation is bioactive in salt water and, as in previous studies on functionalized copper ENMs, that effects are more pronounced at the whole animal than the biochemical level.

Developmental staging and salinity tolerance in embryos of the delta smelt, Hypomesus transpacificus

Delta smelt (Hypomesus transpacificus) is a critically endangered species endemic to the San Francisco Bay Delta (SFBD). Important for the conservation of this species is understanding the physiological and ecological impacts contributing to their population decline, and current studies lack information on embryonic development. Changes in patterns of salinity across the SFBD may be a particularly important environmental stressor contributing to the recruitment and survival of the species. Throughout their ontogeny, delta smelt may exhibit unique requirements and tolerances to environmental conditions including salinity. Here, we describe 22 stages of embryonic development of delta smelt that characterize early differentiation from the fertilized egg until hatching, allowing the identification of critical morphological features unique to this species. Additionally, we investigated aspects of physiological tolerance to environmental salinity during development. Embryos survived incubation at salinity treatments between 0.4 and 20 ppt, yet had lower hatch success at higher salinities. Prior to hatching, embryos exposed to higher salinities had increased osmolalities and reduced fractions of yolk implying that the elevated external salinity altered the physiology of the embryo and the environment internal to the chorion. Lastly, egg activation and fertilization appear to also be impacted by salinity. Altogether, we suggest that any potential tolerance to salinity during embryogenesis, a common feature in euryhaline teleost species, impacts life cycle transitions into, and out of, embryonic development. Results from this investigation should improve conservation and management practices of this species and further expand our understanding of the intimate relationship between an embryo and its environment.

Na+-K+-ATPase and nka genes in spotted sea bass (Lateolabrax maculatus) and their involvement in salinity adaptation

Euryhaline teleosts can survive in a wide salinity range via alteration of the molecular mechanisms to maintain internal ionic and osmotic balance in osmoregulatory organs such as gill，kidney and intestine. Na+/K+-ATPase (NKA), plays a crucial role in sustaining intracellular homeostasis and is characterized by association of multiple isoforms of α- and β-subunits. To gain insight into the potential function of nka genes in salinity adaptation, 5 nkaα genes (nkaα1a, nkaα1b, nkaα2, nkaα3a, nkaα3b) and 7 nkaβ genes (nkaβ1a, nkaβ1b, nkaβ2a, nkaβ2b, nkaβ3a, nkaβ3b and nkaβ4) were identified from transcriptomic and genomic databases of Lateolabrax maculatus. The annotation and evolutionary footprint of these nka genes was revealed via the analysis of phylogenetic tree, gene synteny, copy numbers, exon-intron structures and motif compositions. The expressions of 12 nka genes in spotted sea bass was tested in ten tissues (kidney, gonad, stomach, intestine, gill, muscle, heart, spleen, liver and brain) and 6 genes (nkaα1a, nkaα1b, nkaα3a, nkaα3b, nkaβ1b and nkaβ2a) showed high expression in osmoregulatory organs. Furthermore, the responses of NKA and potential salinity-sensitive nka genes were examined under different salinity treatment (0 ppt, 12 ppt, 30 ppt, 45 ppt). Results showed that the enzyme activity of NKA was highest in gill and exhibited salinity dependent variation, with the highest activity identified in 45 ppt. Different nkaα/β-isoforms showed their diverse responses to salinity changes and the expression of nka genes including nkaα1a, nkaα3b, nkaβ1b in gill, nkaα3a in kidney and nkaβ2a in intestine were transcriptionally regulated by altered salinity. Notably, the expression patterns of nkaα1a and nkaβ1b in gill showed similar variation trend with NKA activity, suggesting that nkaα1a/β1b could be the major function isoforms involved in primary ion transport during salinity adaptation. Our results provided insights into the roles of nkas in osmotic regulation and a theoretical basis for future studies that focus on detailed molecular mechanisms in salinity adaptation of euryhaline teleosts.

14-3-3 gene family in spotted sea bass (Lateolabrax maculatus): Genome-wide identification, phylogenetic analysis and expression profiles after salinity stress

The tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation (14-3-3) proteins are a group of highly conserved homologous and heterologous proteins involved in a wild range of physiological processes, including the regulation of many molecular phenomena under different environmental salinities. In this study, we identified eleven 14-3-3 genes from the spotted sea bass (Lateolabrax maculatus) genome and transcriptomic databases and verified their identities by conducting phylogenetic, syntenic and gene structure analyses. The spotted sea bass 14-3-3 genes are highly conserved based on sequence alignment, conserved domains and motifs, and tertiary structural feature. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 14-3-3 genes in gill of spotted sea bass under normal physiological conditions indicated that the expression level of 14-3-3 zeta was the highest among tested genes, followed by 14-3-3 theta. Furthermore, expression profiles of 14-3-3 genes in gill tissue (in vivo and in vitro) indicated that the 14-3-3 zeta and 14-3-3 theta genes were significantly induced by different environmental salinities in spotted sea bass, suggesting their potential involvement in response to salinity challenge. Our findings may lay the foundation for future functional studies on the 14-3-3 gene family in euryhaline teleosts.

Evidence for a role of arginine vasotocin receptors in the gill during salinity acclimation by a euryhaline teleost fish.

The nonapeptide arginine vasotocin (AVT) regulates osmotic balance in teleost fishes, but its mechanisms of action are not fully understood. Recently, it was discovered that nonapeptide receptors in teleost fishes are differentiated into two V1a-type, several V2-type, and two isotocin (IT) receptors, but it remains unclear which receptors mediate AVT's effects on gill osmoregulation. Here, we examined the role of nonapeptide receptors in the gill of the euryhaline Amargosa pupfish (Cyprinodon nevadensis amargosae) during osmotic acclimation. Transcripts for the teleost V1a-type receptor v1a2 were upregulated over fourfold in gill 24 h after transferring pupfish from 7.5 ppt to seawater (35 ppt) or hypersaline (55 ppt) conditions and downregulated after transfer to freshwater (0.3 ppt). Gill transcripts for the nonapeptide degradation enzyme leucyl-cystinyl aminopeptidase (LNPEP) also increased in fish acclimating to 35 ppt. To test whether the effects of AVT on the gill might be mediated by a V1a-type receptor, we administered AVT or a V1-type receptor antagonist (Manning compound) intraperitoneally to pupfish before transfer to 0.4 ppt or 35 ppt. Pupfish transferred to 35 ppt exhibited elevated gill mRNA abundance for cystic fibrosis transmembrane conductance regulator (cftr), but that upregulation diminished under V1-receptor inhibition. AVT inhibited the increase in gill Na+/Cl- cotransporter 2 (ncc2) transcript abundance that occurs following transfer to hypoosmotic environments, whereas V1-type receptor antagonism increased ncc2 mRNAs even without a change in salinity. These findings indicate that AVT acts via a V1-type receptor to regulate gill Cl- transport by inhibiting Cl- uptake and facilitating Cl- secretion during seawater acclimation.

Benzocaine-induced stress in the euryhaline teleost, Centropomus parallelus and its implications for anesthesia protocols

The use of anesthetic in fish farming is a traditional practice which aims to reduce the stress caused by transport and handling. However, anesthesia-induction protocols are commonly established and implemented without proper physiological/behavioral evaluation. Additionally, concentration and time of exposure to the anesthetic are often set without considering species-specific responses. The fat snook (Centropomus parallelus) is a fish with great potential for aquaculture. Given its remarkable euryhalinity, it can grow in fresh- or seawater. Most studies on fat snook anesthesia tested natural compounds (essential oils) instead of traditional anesthetics. However, the use of benzocaine is much more common in the commercial sector, as it is easy to obtain and of relatively low cost. The present study aimed at analyzing the effects benzocaine exposure on glucose and cortisol plasma levels (two traditional stress markers in teleost fish), as well as on plasma osmolality, chloride and magnesium, (indicators of osmo-ionic allostasis) in animals acclimated to different salinities. Results showed that while osmo-ionic allostasis was strictly maintained across the treatments, time of anesthesia had a strong positive relationship to plasma cortisol and glucose, regardless the salinity of exposure and acclimation. The results are discussed as they relate to anesthesia protocols and how stress response generated by time of anesthesia may challenge farming flexibility.

Dynamic expression of vasotocin and isotocin receptor genes in the marbled eel (Anguilla marmorata) following osmotic challenges

To examine the physiological roles of arginine vasotocin receptor (AVTR) and isotocin receptor (ITR) in osmoregulation of a euryhaline teleost, the marbled eel (Anguilla marmorata), three different genes coding for AVTRV1a2, AVTRV2 and ITR were cloned by screening an A. marmorata cDNA library. These receptors were expressed differentially and ubiquitously in the eight tissues we examined. The changes in mRNA expression levels of AVTRV1a2, AVTRV2, and ITR were assessed in a time-course study following salinity transfer from fresh water (FW, 0‰) to fresh water (FW, 0‰), brackish water (BW, 10‰) or saline water (SW, 25‰). When eels were transferred to BW, mRNA levels underwent an adaptive period, from 0 to 24 h, and a chronic regulatory period, starting at 24 h after transfer. In the adaptive period, the relative mRNA expression of AVTRV1a2, AVTRV2, and ITR increased in BW. But after this adaptive period, the mRNA levels of the three genes were significantly decreased compared to FW (control group, 0 h). The mRNA expression levels of AVTRV1a2, AVTRV2 and ITR were low in SW. The protein level of AVTRV1a2, a key protein in the brain, was also investigated and found to be consistent with mRNA results. Our results indicated that the nonapeptide receptor system may play a role in the acute stress response induced by hyper-osmotic challenge in marbled eels.

An assay of optimal cytochrome c oxidase activity in fish gills

Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50–100 μg fish gill homogenate in conjunction with 75–100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish.