Transport and Barrier Functions in Rainbow Trout Trunk Skin Are Regulated by Environmental Salinity.

The mechanisms underpinning ionic transport and barrier function have been relatively well characterised in amphibians and fish. In teleost fish, these processes have mostly been characterised in the gill and intestine. In contrast, these processes remain much less clear for the trunk skin of fish. In this study, we measured barrier function and active transport in the trunk skin of the rainbow trout, using the Ussing chamber technique. The effects of epithelial damage, skin region, salinity, and pharmacological inhibition were tested. Skin barrier function decreased significantly after the infliction of a superficial wound through the removal of scales. Wound healing was already underway after 3 h and, after 24 h, there was no significant difference in barrier function towards ions between the wounded and control skin. In relation to salinity, skin permeability decreased drastically following exposure to freshwater, and increased following exposure to seawater. Changes in epithelial permeability were accompanied by salinity-dependent changes in transepithelial potential and short-circuit current. The results of this study support the idea that barrier function in rainbow trout trunk skin is regulated by tight junctions that rapidly respond to changes in salinity. The changes in transepithelial permeability and short circuit current also suggest the presence of an active transport component. Immunostaining and selective inhibition suggest that one active transport component is an apical V-ATPase. However, further research is required to determine the exact role of this transporter in the context of the trunk skin.

Calcium mobilisation following shell damage in the Pacific oyster, Crassostrea gigas.

Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to (45)Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.

Stunning fish with CO2 or electricity: contradictory results on behavioural and physiological stress responses.

Studies that address fish welfare before slaughter have concluded that many of the traditional systems used to stun fish including CO2 narcosis are unacceptable as they cause avoidable stress before death. One system recommended as a better alternative is electrical stunning, however, the welfare aspects of this method are not yet fully understood. To assess welfare in aquaculture both behavioural and physiological measurements have been used, but few studies have examined the relationship between these variables. In an on-site study aversive behaviours and several physiological stress indicators, including plasma levels of cortisol and ions as well as blood physiological variables, were compared in Arctic char (Salvelinus alpinus) stunned with CO2 or electricity. Exposure to water saturated with CO2 triggered aversive struggling and escape responses for several minutes before immobilization, whereas in fish exposed to an electric current immobilization was close to instant. On average, it took 5 min for the fish to recover from electrical stunning, whereas fish stunned with CO2 did not recover. Despite this, the electrically stunned fish had more than double the plasma levels of cortisol compared with fish stunned with CO2. This result is surprising considering that the behavioural reactions were much more pronounced following CO2 exposure. These contradictory results are discussed with regard to animal welfare and stress physiological responses. The present results emphasise the importance of using an integrative and interdisciplinary approach and to include both behavioural and physiological stress indicators in order to make accurate welfare assessments of fish in aquaculture.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar.

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmon Salmo salar. Emphasis was placed on Na(+) , K(+) -ATPase (NKA) and Na(+) , K(+) , Cl(-) co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na(+) , Cl(-) co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Plasma growth hormone-binding protein levels in Atlantic salmon Salmo salar during smoltification and seawater transfer.

Specific growth hormone (GH)-binding protein (Ghbp) was purified from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss plasma with immunoprecipitation and characterized in cross-linking studies using autoradiography and western blots. The size of the Ghbp was estimated to be c. 53 kDa. A radioimmunoassay was established to measure Ghbp in salmonids, using antibodies specific against the extracellular segment of the S. salar growth hormone receptor 1 (grh1; GenBank AY462105). Plasma Ghbp levels were measured in S. salar smolts in fresh water and after transfer to seawater (SW; experiments 1 and 2), and in post-smolts kept at different salinities (0, 12, 22 and 34) for 3 months (experiment 3). A transient increase in plasma Ghbp, which lasted for 1 month or less, was noted in smolts after transfer to SW. Concomitantly, plasma GH and gill Na(+) -K(+) -ATPase activity increased during smoltification (in experiment 2). No difference in plasma Ghbp was evident between post-smolts kept at different salinities, although the fish kept at salinity 34 had higher plasma GH than the group kept at salinity 22 and higher hepatic ghr1 expression than post-smolts kept at salinity 12. This suggests that plasma Ghbp regulation may respond to salinity changes in the short term. The lack of correlation between Ghbp, plasma GH and hepatic ghr1 expression in the long-term post-smolt experiment indicates that Ghbp levels may be regulated independently of other components of the endocrine GH system in salmonids.

Slow release cortisol implants result in impaired innate immune responses and higher infection prevalence following experimental challenge with infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) parr.

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 µg cortisol g(-1) body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples. Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr.

Disturbance of the intestinal mucosal immune system of farmed Atlantic salmon (Salmo salar), in response to long-term hypoxic conditions.

The gastrointestinal (GI) tract has many important biological functions. One is to serve as a barrier between the fish and the external environment. A decreased physical barrier function of the intestine may lead to increased inflow of luminal content and subsequent activation of the intestinal mucosal immune system. This activation is governed by the ability of various compounds to induce cytokine release and immune cell activity, leading to an immune response. In mammals, the impact of stress on the intestinal barrier is well documented and results in increased intestinal permeability and thus increased stimulation of the mucosal immune system. Fish reared in sea cages may at times be exposed to unfavourable environmental conditions leading to chronic stress and disturbed intestinal integrity. This change in permeability may increase the exposure of the mucosal immune system to activating compounds. In the present study, the effect of a prolonged stress on the intestinal mucosal immune system of fish is therefore addressed. Atlantic salmon were exposed to low levels (50%) of dissolved oxygen (DO) for 6-7 weeks in consecutive experiments performed at 8 and 16 °C. Immune parameters were assessed in terms of mRNA expression of the key cytokines, interleukin-1ß (IL-1ß), IL-8, IL-10, interferon-γ (IFNγ) and transforming growth factor-ß (TGFß) as well as the immune regulatory inhibitor of nuclear factor κB (IκB). In the experiment at 8 °C also mucosal neutrophil infiltration was monitored. Subjecting the fish to low DO levels at 8 °C resulted in an increased mucosal neutrophil infiltration together with a down-regulation of IκB. At the higher temperature, 16 °C, low DO levels created decreased expression of the pro-inflammatory cytokine IL-1ß in both intestinal regions as well as an increased expression of IL-10 in the proximal intestine. These results suggest that husbandry conditions in sea cages with DO levels as low as 50% clearly affects the intestinal mucosal immune system and results in a chronic inflammation. Moreover, the effects of low DO levels on the immune factors examined were more pronounced in the 16 °C experiment suggesting additive effects of high temperatures.

The effect of hyperoxygenation and reduced flow in fresh water and subsequent infectious pancreatic necrosis virus challenge in sea water, on the intestinal barrier integrity in Atlantic salmon, Salmo salar L.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.

The involvement of Aeromonas salmonicida virulence factors in bacterial translocation across the rainbow trout, Oncorhynchus mykiss (Walbaum), intestine.

The pathogenic bacterium Aeromonas salmonicida is the causative agent of furunculosis, a lethal disease in salmonids. The mode of lateral transmission has not been conclusively defined, but A. salmonicida is able to translocate across the intestinal epithelium of salmonids, making the intestinal route a probable candidate. This study investigated some of the virulence mechanisms used by the bacteria to promote translocation. Intestinal segments were placed in modified Ussing chambers to investigate epithelial functions during exposure to bacterial factors. The factors were: extracellular products (ECP), lipopolysaccharide (LPS) or live or heat-inactivated A. salmonicida. Fluorescein isothiocynate (FITC)-labelling enabled detection of translocated bacteria by fluorometry. Live A. salmonicida translocated to a greater degree than heat-inactivated bacteria, suggesting that the bacteria utilize a heat sensitive surface-bound virulence factor which promotes translocation. The epithelium was negatively affected by ECP, manifested as decreased net ion transport, indicating a disturbance in ion channels or cell metabolism. LPS did not affect the epithelium in vitro when administered on the luminal side of the intestinal segment, but significantly increased epithelial translocation of fluorescent bacterial-sized microspheres when administered on the serosal side. This is suggested to be caused by increased transcellular transport, as the paracellular permeability was unaffected indicating maintained epithelial integrity.