From Freshwater Research Articles

Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia

In estuarine environments, euryhaline fish maintain a narrow range of internal osmolality despite daily changes in environmental salinity that can range from fresh water (FW) to seawater (SW). The capacity of euryhaline fish to maintain homeostasis in a range of environmental salinities is primarily facilitated by the neuroendocrine system. One such system, the hypothalamic-pituitary-interrenal (HPI) axis, culminates in the release of corticosteroids such as cortisol into circulation. Cortisol functions as both a mineralocorticoid and glucocorticoid in fish because of its roles in osmoregulation and metabolism, respectively. The gill, a key site for osmoregulation, and the liver, the primary storage site for glucose, are known targets of cortisol’s actions during salinity stress. While cortisol facilitates acclimation to SW environments, less is known on its role during FW adaptation. In this study, we characterized the responses of plasma cortisol, mRNA expression of pituitary pro-opiomelanocortin (pomc), and mRNA expression of liver and gill corticosteroid receptors (gr1, gr2, and mr) in the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity challenges. Specifically, tilapia were subjected to salinity transfer regimes from steady-state FW to SW, SW to FW (experiment 1) or steady state FW or SW to tidal regimen (TR, experiment 2). In experiment 1, fish were sampled at 0 h, 6 h, 1, 2, and 7 d post transfer; while in experiment 2, fish were sampled at day 0 and day 15. We found a rise in pituitary pomc expression and plasma cortisol following transfer to SW while branchial corticosteroid receptors were immediately downregulated after transfer to FW. Moreover, branchial expression of corticosteroid receptors changed with each salinity phase of the TR, suggesting rapid environmental modulation of corticosteorid action. Together, these results support the role of the HPI-axis in promoting salinity acclimation, including in dynamically-changing environments.

Transcriptomic Modulation Reveals the Specific Cellular Response in Chinese Sea Bass (Lateolabrax maculatus) Gills under Salinity Change and Alkalinity Stress.

Salinity and alkalinity are among the important factors affecting the distribution, survival, growth and physiology of aquatic animals. Chinese sea bass (Lateolabrax maculatus) is an important aquaculture fish species in China that can widely adapt to diverse salinities from freshwater (FW) to seawater (SW) but moderately adapt to highly alkaline water (AW). In this study, juvenile L. maculatus were exposed to salinity change (SW to FW) and alkalinity stress (FW to AW). Coordinated transcriptomic responses in L. maculatus gills were investigated and based on the weighted gene co-expression network analysis (WGCNA), 8 and 11 stress-responsive modules (SRMs) were identified for salinity change and alkalinity stress, respectively, which revealed a cascade of cellular responses to oxidative and osmotic stress in L. maculatus gills. Specifically, four upregulated SRMs were enriched with induced differentially expressed genes (DEGs) for alkalinity stress, mainly corresponding to the functions of "extracellular matrix" and "anatomical structure", indicating a strong cellular response to alkaline water. Both "antioxidative activity" and "immune response" functions were enriched in the downregulated alkaline SRMs, which comprised inhibited alkaline specific DEGs, revealing the severely disrupted immune and antioxidative functions under alkalinity stress. These alkaline-specific responses were not revealed in the salinity change groups with only moderately inhibited osmoregulation and induced antioxidative response in L. maculatus gills. Therefore, the results revealed the diverse and correlated regulation of the cellular process and stress response in saline-alkaline water, which may have arisen through the functional divergence and adaptive recruitment of the co-expression genes and will provide vital insights for the development of L. maculatus cultivation in alkaline water.

Effects of Osmotic Stress on the mRNA Expression of prl, prlr, gr, gh, and ghr in the Pituitary and Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii.

In euryhaline teleost black porgy, Acanthopagrus schlegelii, the glucocorticoid receptor (gr), growth hormone receptor (ghr), prolactin (prl)-receptor (prlr), and sodium-potassium ATPase alpha subunit (α-nka) play essential physiological roles in the osmoregulatory organs, including the gill, kidney, and intestine, during osmotic stress. The present study aimed to investigate the impact of pituitary hormones and hormone receptors in the osmoregulatory organs during the transfer from freshwater (FW) to 4 ppt and seawater (SW) and vice versa in black porgy. Quantitative real-time PCR (Q-PCR) was carried out to analyze the transcript levels during salinity and osmoregulatory stress. Increased salinity resulted in decreased transcripts of prl in the pituitary, α-nka and prlr in the gill, and α-nka and prlr in the kidney. Increased salinity caused the increased transcripts of gr in the gill and α-nka in the intestine. Decreased salinity resulted in increased pituitary prl, and increases in α-nka and prlr in the gill, and α-nka, prlr, and ghr in the kidney. Taken together, the present results highlight the involvement of prl, prlr, gh, and ghr in the osmoregulation and osmotic stress in the osmoregulatory organs (gill, intestine, and kidney). Pituitary prl, and gill and intestine prlr are consistently downregulated during the increased salinity stress and vice versa. It is suggested that prl plays a more significant role in osmoregulation than gh in the euryhaline black porgy. Furthermore, the present results highlighted that the gill gr transcript's role was solely to balance the homeostasis in the black porgy during salinity stress.

Significance of sea entry pathway of chum salmon Oncorhynchus keta fry, inferred from the differential expressions of Na+,K+-ATPase α-subunit genes in the gills

Na+,K+-ATPase (NKA) α-subunit 1a (α1a) and 1b (α1b) gene expressions in the gills are changeable in response to ambient salinity in a few salmonids. In this study, the expressions were compared among ambient salinities and used to infer sea entry migration of chum salmon Oncorhynchus keta fry. The expression of α1a decreased from the 2 days after seawater (SW) transfer from freshwater (FW) and was significantly lower in SW-acclimated fry than that in FW-fry. On the other hand, the expression of α1b peaked on the first to second day after SW transfer and then settled to a level 2-fold higher than in FW-fry. In fry caught in the waterfronts of the beaches, the expression levels were quite similar to those on the first and second days after SW transfer, whereas, in fry caught off beach, the expressions were identical to those of SW-acclimated fry. These suggest that fry adapt to SW with moving along the shoal in the bay, and move to off beach after completing SW adaptation. One of the physiological significances in a usage of waterfront may be to transform the gills to SW type. Only fry on the 2 days after SW transfer failed to exhibit condition factor-dependency of burst swimming, probably due to physiological perturbation, which may be related to poor predation avoidance. The physiological approach used in this study inferred sea entry migration of fry; furthermore, it shows the possible significance of adaptation to SW in the shoal is to reduce predation risk.

Age-Dependent Decline in Salinity Tolerance in a Euryhaline Fish.

Euryhaline teleost fish are characterized by their ability to tolerate a wide range of environmental salinities by modifying the function of osmoregulatory cells and tissues. In this study, we experimentally addressed the age-related decline in the sensitivity of osmoregulatory transcripts associated with a transfer from fresh water (FW) to seawater (SW) in the euryhaline teleost, Mozambique tilapia, Oreochromis mossambicus. The survival rates of tilapia transferred from FW to SW were inversely related with age, indicating that older fish require a longer acclimation period during a salinity challenge. The relative expression of Na+/K+/2Cl− cotransporter 1a (nkcc1a), which plays an important role in hyposmoregulation, was significantly upregulated in younger fish after SW transfer, indicating a clear effect of age in the sensitivity of branchial ionocytes. Prolactin (Prl), a hyperosmoregulatory hormone in O. mossambicus, is released in direct response to a fall in extracellular osmolality. Prl cells of 4-month-old tilapia were sensitive to hyposmotic stimuli, while those of >24-month-old fish did not respond. Moreover, the responsiveness of branchial ionocytes to Prl was more robust in younger fish. Taken together, multiple aspects of osmotic homeostasis, from osmoreception to hormonal and environmental control of osmoregulation, declined in older fish. This decline appears to undermine the ability of older fish to survive transfer to hyperosmotic environments.

Hypothermal stress-induced salinity-dependent oxidative stress and apoptosis in the livers of euryhaline milkfish, Chanos chanos

Water temperature is one of the key environmental factors affecting the physiological responses of fish, hence it becomes a significant concern for aquaculture fisheries. The euryhaline milkfish, Chanos chanos, one of the major aquaculture species in Southeast Asia, can be cultured in ponds of different salinities ranging from fresh water (FW) to sea water (SW). Being a tropical species, high mortality occurs in cultured milkfish after a cold snap in winter, leading to a huge economic loss. In the present study, we studied salinity effects on mechanisms underlying hypothermal tolerance in milkfish using various molecular and physiological approaches. At low temperatures, FW milkfish were found to have poorer cold-tolerance ability and more oxidative stress than SW milkfish. In addition, milkfish hepatocytes exhibited apoptosis induced via an extrinsic or intrinsic pathway, indicating the source of oxidative stress. This study further revealed that under low-temperature acclimation, more hepatocytes were shrunk and showed positive response to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in FW milkfish than in SW milkfish. The caspase-3 activity was up-regulated, which, in turn induced apoptosis via an extrinsic pathway in the hepatocytes of FW milkfish under hypothermal stress. Although the enzymatic antioxidant mechanisms were up-regulated to neutralize oxidative stress, apoptosis was still observed in the hepatocytes of FW milkfish under hypothermal stress. On the other hand, the hepatocytes of SW milkfish under identical hypothermal conditions showed no redox imbalance and apoptotic responses. Overall, this study suggests a hypothermal FW environment induces oxidative stress and apoptosis in hepatocytes and impairs liver functions that may lead to cold intolerance in milkfish.

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.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na+/K+-ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H+-ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na+ uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.

Immunohistochemical characterization and change in location of branchial ionocytes after transfer from freshwater to seawater in the euryhaline obscure puffer, Takifugu obscurus.

The obscurepuffer Takifugu obscurus is a euryhaline fish species suitable for studying the molecular mechanism of osmoregulation. The distributional changes of branchial ionocytes were detected following the transfer from freshwater (FW) to seawater (SW) based on two main ion transporters, Na+/K+-ATPase (NKA) and Na+/K+/ 2Cl- cotransporter 1 (NKCC1). The mRNA and protein expression levels of NKA and NKCC1 in the gills all increased rapidly in the first four days after transfer to SW. Double immunofluorescence staining showed that NKCC1 and NKA were colocalized in the branchial ionocytes and the immunoreaction of NKCC1 was stronger after transfer. Moreover, following transfer to SW, the number of lamellar ionocytes in the gills is reduced and the number of filament ionocytes is increased significantly. Taken together, these findings indicated that SW transfer of obscure puffer promotes the changes of distribution, function and size of branchial ionocytes.

A cis‐regulatory Element in Intron 1 Cooperates with the Core Promoter during Salinity Induction of Tilapia Glutamine Synthetase

Mozambique tilapia (O. mossambicus) is a euryhaline fish that tolerates salinity ranging from fresh water (FW) to 120 g/kg (> 3x seawater, SW). Hyperosmotic stress associated with salinity transfer from FW to SW strongly induces glutamine synthetase (GS) transcription in tilapia, including a brain cell line model (OmB cells). In this study we cloned the 5′ regulatory sequence (RS) (including 5′‐flanking region and 5′‐UTR) of the O. mossambicus GS gene to identify osmotic response elements that mediate transcriptional induction. Using enhancer trapping, we discovered a novel osmosensitive mechanism that utilizes an unusual enhancer cassette in intron 1 of the GS gene. This cassette includes a single copy of a cis‐regulatory element (CRE), the osmolality/salinity‐responsive element 1 (OSRE1). We show that intronic OSRE1 is necessary but not sufficient for intron 1‐mediated hyperosmotic GS induction. Furthermore, intron 1 and its interaction with the GS core promoter complex are also necessary for hyperosmotic GS induction. We propose a novel model for hyperosmotic intron 1‐mediated induction of the O. mossambicus GS gene that is based on the interaction of an OSRE1 enhancer, intron 1, and the core GS promotor.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).

Regulation of 14-3-3β/α gene expression in response to salinity, thermal, and bacterial stresses in Siberian sturgeon (Acipenser baeri).

The 14-3-3 proteins are a family of widely expressed acidic proteins, which are involved in the regulation of many biological processes of animals. However, no research regarding 14-3-3 has been described in sturgeon to date, one of the most primitive Actinopterygii species. Here, we identified the first 14-3-3 gene from Siberian sturgeon (Acipenser baeri), named Ab14-3-3β/α (GenBank Accession No. KY094076.1). The cDNA of Ab14-3-3β/α is 1212bp in length, containing a 5'-untranslated region (UTR) of 82bp, a 3'UTR of 392bp, and an open reading frame (ORF) of 738bp, encoding a polypeptide of 245 amino acids which contains a 14-3-3 homologs domain (PF00244). Phylogenetic analysis showed that the 14-3-3 gene product from Acipenser baeri is a counterpart of vertebrate 14-3-3β/α. The deduced Ab14-3-3β/α protein shares high identities of 46.5-95.5% with the homologs of other species. Ab14-3-3β/α mRNA was constitutively expressed in all examined tissues, with high expression levels in the blood and gill. Furthermore, the expression level of Ab14-3-3β/α mRNA increased significantly in the gill at 1h under acute salinity shock by transfer of Siberian sturgeons from fresh water (FW) to 15ppt. In fish subjected to a high temperature (31°C), Ab14-3-3β/α showed a significant upregulation in the liver at 3h compared with the control group (24°C). A 4.85-fold increase of Ab14-3-3β/α expression in the spleen of Siberian sturgeon was observed at 24h following Aeromonas hydrophila challenge. Collectively, these results indicated that Ab14-3-3β/α might play a certain role in sturgeon in response to some environmental stresses and bacterial challenge.

Characterization of smoltification in the Tasmanian strain of Atlantic salmon (Salmo salar) in recirculation and flow-through systems

This study examined morphological, physiological and molecular indicators of smoltification in Atlantic salmon (Salmo salar) juveniles in a flow-through (FT) and recirculating aquaculture system (RAS). Fish were exposed to 24-h light to initiate smoltification, for 5 (FT) and 7 (RAS) weeks prior to transfer from freshwater (FW) to seawater (SW) and were sampled weekly preceding and following SW transfer. Mass, length, condition factor, plasma chloride, gill Na+/K+-ATPase (NKA) activity and expression of salinity-specific isoforms of NKA mRNA transcripts were monitored. Fish raised in FT had significantly lower specific growth rate (SGR) in FW than in SW and showed a significant 5-6-fold increase in gill NKA activity, and high plasma chloride levels after transfer to SW. These fish also exhibited no significant reduction in relative mRNA expression of NKAα1a in FW but a sharp significant downregulation post-SW transfer. No significant increase in NKAα1b was seen until week 8 (3 weeks post-SW transfer). The log2 ratio of NKAα1b to NKAα1a showed a significant 8-fold increase throughout the study. Fish raised in the RAS had significantly higher SGR in FW than SW, and showed significantly higher plasma chloride in saltwater challenged fish compared to the freshwater control at all weeks during the FW phase. Fish had a 50% higher initial NKA activity than in FT, increasing significantly 2-3-fold and showed an immediate down-regulation of NKAα1a after exposure to 24-hr light in FW, and a further reduction after SW-transfer. There was no significant increase in NKAα1b in the RAS-raised fish for the duration of the study, and there was a significant 8-fold increase in log2 ratio of NKAα1b to NKAα1a. Whilst there were too many varying factors to statistically compare hatchery type in this study, it's evident that there are potentially system-related effects worthy of future investigation.

A comparison of infectious agents between hatchery-enhanced and wild out-migrating juvenile chinook salmon (Oncorhynchus tshawytscha) from Cowichan River, British Columbia

Infectious diseases are likely contributing to large-scale declines in chinook salmon stocks in the Pacific Northwest, but the specific agents and diseases involved, and the prevalences in migratory salmon, are mostly unknown. We applied a high-throughput microfluidics platform to screen for 45 infectious agents in 556 out-migrating juvenile chinook salmon, collected from freshwater (FW) and saltwater (SW) locations in the Cowichan River system on Vancouver Island, Canada, during 2014. Nineteen agents (5 bacterial, 2 viral, and 12 parasitic) were detected, with prevalences ranging from 0.2% to 57.6%. Co-infections between Candidatus Branchiomonas cysticola Toenshoff, Kvellestad, Mitchell, Steinum, Falk, Colquhoun & Horn, 2012, Paranucleospora theridion Nylund, Nylund, Watanabe, Arnesen & Kalrsbakk, 2010, and gill chlamydia, all associated with gill disease, were observed in SW samples. We detected agents known to cause large-scale mortalities in Pacific salmon ( Ceratonova shasta (Noble, 1950), Parvicapsula minibicornis Kent, Whitaker & Dawe, 1977), and agents only recently reported in Pacific salmon in BC ( Ca. B. cysticola, P. theridion, Facilispora margolisi Jones, Prosperi-Porta & Kim, 2012 and Parvicapsula pseudobranchicola Karlsbakk, Saether, Hostlund, Fjellsoy & Nylund, 2002). Wild and hatchery fish were most divergent in agent profiles in FW, with higher agent diversity in wild fish. Differences in prevalence largely dissipated once they converged in the marine environment, although hatchery fish may be infected by a greater diversity of agents sooner after ocean entry by virtue of their more rapid migration from nearshore to offshore environments.

Multistage wet lipid extraction from fresh water stressed Neochloris oleoabundans slurry – Experiments and modelling

Algae are considered an important renewable feedstock for lipid extraction to produce biofuels. Algae strain Neochloris oleoabundans used in this research can yield a high lipid content under stressed conditions. N-ethyl butylamine (EBA) as a switchable solvent has previously shown outstanding performance on energy efficient lipid extraction from non-broken wet algae slurry. In this work, a model was developed that describes the equilibrium state of lipid extraction from fresh water (FW)-stressed Neochloris oleoabundans algae slurry using EBA as solvent. When assuming that the cell interior is almost completely filled with the solvent phase during extraction, the model estimated extraction yields showed good agreement with those obtained in experiments. The developed model can predict the amount of crude lipid being recovered from any stage of a multistage extraction process.

A sodium binding system alleviates acute salt stress during seawater acclimation in eels

BackgroundTeleosts transiting from freshwater (FW) to seawater (SW) environments face an immediate osmotic stress from ion influxes and water loss, but some euryhaline species such as eels can maintain a stable plasma osmolality during early SW exposure. The time course changes in the gene expression, protein abundance, and localization of key ion transporters suggested that the reversal of the ion transport systems was gradual, and we investigate how eels utilize a Na-binding strategy to slow down the ion invasion and complement the transporter-mediated osmoregulation.ResultsUsing an electron probe micro-analyzer, we localized bound Na in various eel tissues in response to SW transfer, suggesting that the Na-binding molecules were produced to sequester excess ionic Na+ to negate its osmotic potential, thus preventing acute cellular dehydration. Mucus cells were acutely activated in digestive tract, gill, and skin after SW transfer, producing Na-binding molecule-containing mucus layers that fence off high osmolality of SW. Using gel filtration HPLC, some molecules at 18 kDa were found to bind Na in the luminal secretion of esophagus and intestine, and higher binding was associated with SW transfer. Transcriptome and protein interaction results indicated that downregulation of Notch and β-catenin pathways, and dynamic changes in TGFβ pathways in intestine were involved during early SW transition, supporting the observed histological changes on epithelial desquamation and increased mucus production.ConclusionsThe timing for the activation of the Na-binding mechanism to alleviate the adverse osmotic gradient was temporally complementary to the subsequent remodeling of branchial ionocytes and transporting epithelia of the digestive tract. The strategy to manipulate the osmotic potential of Na+ by specific binding molecules is similar to the osmotically inactive Na described in human skin and muscle. The Na-binding molecules provide a buffer to tolerate the salinity changes, which is advantageous to the estuary and migrating fishes. Our data pave the way to identify this unknown class of molecules and open a new area of vertebrate osmoregulation research.

Immunolocalization of Na+ /K+ -ATPase, Na+ /K+ /2Cl− co-transporter (NKCC) and mRNA expression of Na+ /K+ -ATPase α-subunit during short-term salinity transfer in the gills of Persian sturgeon (Acipenser persicus , Borodin, 1897) juveniles

The study tests the physiological responses of Persian sturgeon, Acipenser persicus, during the abrupt release of juveniles from freshwater (FW) into brackish waters (BW = 11‰) of the Caspian Sea. Fish weight at release was 2-3 g (2.55 ± 0.41 g; 8.8 ± 0.58 cm TL). Totals of 160 individuals were randomly distributed into four fiber-glass aerated tanks (volume 60-L). Two tanks served as controls (FW groups), and two as exposure tanks for BW (Caspian Sea water = CSW). Fish were sampled at 0, 3, 6, 12, 24, 48 and 96 hr after abrupt transfer to CSW. Plasma osmolality, immunolocalization of Na+, K+ -ATPase (NKA) and Na+/K+/2Cl– (NKCC) Co-transporter, NKA activity and the NKA α-subunit mRNA expression were analyzed. Blood osmolality of fish transferred from FW to CSW increased significantly within hours post-transfer (p < .05) and remained at a high level for up to 96 hr. Immunolocalization of NKCC indicated co-localization with NKA in the chloride cells in the gill epithelium. A partial sequence of the NKA α-subunit (632 bp) is described. Its expression levels were up-regulated at 12 and 48 hr following salinity transfer (p < .05). However, NKA activity sharply increased in CSW specimens by almost 2.8-fold (p < .05) between 48 and 96 hr after transfer. Gill NKCC co-transporter abundance increased, coinciding with increased gill NKA activity. The increased activity of NKCC during salt excretion in CSW may lead to an influx of Na+ into the chloride cells. Consequently, NKA activity increases to maintain intracellular Na+ homeostasis.

Water repellency reduction using soil heating in infiltration ponds of a wastewater soil aquifer treatment (SAT)

AbstractReuse of treated wastewater (TWW) for irrigation in agriculture is a common alternative water source in arid regions suffering from fresh water (FW) shortage. Soil aquifer treatment (SAT) is frequently used for advanced TWW purification. Infiltration rates of water through the soil can decrease as a consequence of organic matter (OM) accumulation and the consequent induction of water repellency. In this research, soil heating to high temperatures was examined for its efficiency in OM content reduction and increased infiltration. In a laboratory experiment conducted using a hand burner and a combustion oven, water repellency was found to be reduced following increased heating temperature or heating duration, directly resulting in reduction of OM level. Chemical analysis of OM extracted from the heat treated soils revealed reduction in hydrophobic substances as a consequence of increased temperatures by soil heating and heating duration. In model ponds built to simulate large infiltration basins, OM content was reduced as a result of intensive and moderate soil heating in comparison to the untreated pond. However, no reduction in water repellency and only slight changes in infiltration rate were found as a consequence of soil heating. The differences in results obtained in the laboratory and the field experiments were probably related to the fact that the soil in the field experiment has been continuously reloaded with TWW containing OM.

Claudin-10 isoform expression and cation selectivity change with salinity in salt-secreting epithelia of Fundulusheteroclitus.

To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from freshwater (FW) to seawater (SW, 35‰) and from SW to hypersaline SW (2SW, 60‰) in a time course with transfer control groups (FW to FW, and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and cldn-10e twofold, whilst cldn-10c and cldn-10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d, and transiently altered cldn-10e abundance, but increased cldn-10c and cldn-10f fourfold. This was coincident with an increased number of single-stranded junctions (observed by transmission electron microscopy). For both salinity transfers, (1) cldn-10e mRNA was acutely responsive (i.e. after 24 h), (2) other responsive cldn-10 isoforms increased later (3-7 days), and (3) cystic fibrosis transmembrane conductance regulator (cftr) mRNA was elevated in accordance with established changes in transcellular Cl- movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and cldn-10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na+>K+>Rb+∼Cs+>Li+ Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes.

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis

Estradiol (E2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis.The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E2 signalling pathway along the BPG axis.Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.

Flexible selection of diversified Na(+)/K(+)-ATPase α-subunit isoforms for osmoregulation in teleosts.

Background and methodsMultiple Na+/K+-ATPase (NKA) α-subunit isoforms express differentially in response to salinity transfer in teleosts but we observed that the isoform nomenclature is inconsistent with the phylogenetic relationship of NKA α-genes. We cloned the catalytic NKA α-subunit isoforms in eels and medaka, analyzed the time course of their expressions in osmoregulatory tissues after transfer from freshwater (FW) to seawater (SW), and performed phylogenetic analyses to deduce an evolutionary scenario that illustrates how various duplication events have led to the current genomic arrangement of NKA α-genes in teleosts.Results and discussionFive and six α-subunits were cloned in eels and medaka respectively. In eels, the commonly-reported α1a and α1b isoforms were absent while the α1c isoform was diversified instead (α1c-1, α1c-2, α1c-3, α2, and α3 in eels). Phylogenetic estimation indicated that the α1a and α1b isoforms from salmon, tilapia, and medaka were generated by independent duplication events and thus they are paralogous isoforms. Re-examination of expression changes of known isoforms after salinity challenge revealed that the isoforms selected as predominant SW-types varied among teleost lineages. Diversification of α1 isoforms occurred by various types of gene duplication, or by alternative transcription among tandem genes to form chimeric transcripts, but there is no trend for more α1 copies in euryhaline species. Our data suggest that the isoform switching between FW (α1a predominates) and SW (α1b predominates) that occurs in salmonids is not universal in teleosts. Instead, in eels, α1c-1 was the major α-subunit upregulated gill, intestine, and kidney in SW. Localization of both NKA mRNA and protein showed consistent upregulation in gill and intestine in SW eels, but not in renal distal and collecting tubules, where low transcript expression levels were accompanied by high protein levels, suggesting a tissue-specific translational regulation that determines and fine-tunes the NKA expression. In medaka, α1b was upregulated in SW in anterior intestine while most other α-subunit isoforms were less responsive to salinity changes.ConclusionBy integrating gene expression and phylogenetic results, we propose that the major NKA α-subunits for SW acclimation were not ancestrally selected, but rather were flexibly determined in lineage-specific fashion in teleosts.