Gills Of Salmon Research Articles

Cortisol regulation of ion transporter mRNA in Atlantic salmon gill and the effect of salinity on the signaling pathway

Based on real-time RT-PCR, analysis of transcripts of selected ion-regulatory proteins (Na(+), K(+)-ATPase alpha1a and alpha1b subunit, Na(+), K(+), 2Cl(-) cotransporter, cystic fibrosis transmembrane conductance regulator (CFTR), and H(+)-ATPase B-subunit), the regulatory role of cortisol and the associated receptor signaling pathway (glucocorticoid (GR) versus mineralocorticoid (MR)) of cortisol was investigated in the salmon gill. Using a gill organ culture technique, the effect of cortisol with and without addition of specific hormone receptor antagonists (RU486 and spironolactone) was analyzed in gills from freshwater (FW) and seawater (SW) acclimated fish. The effect of cortisol was highly dependent on acclimation to salinity. In FW, cortisol stimulated the transcript levels of CFTR-I and Na(+), K(+)-ATPase alpha1a and alpha1b. Addition of RU486 totally abolished the cortisol effects on CFTR-I and Na(+), K(+)-ATPase alpha1b, suggesting that signaling was mediated via GR. Interestingly, both spironolactone and RU486 were able to inhibit the cortisol effect on Na(+), K(+)-ATPase alpha1a indicating a role for both MR and GR in regulation of this target gene. In SW, cortisol increased the transcript levels of CFTR-I, CFTR-II, Na(+), K(+)-ATPase alpha1a and alpha1b, and NKCC. Interestingly, the effect of cortisol on CFTR-I and Na(+), K(+)-ATPase alpha1a was mediated through GR and MR respectively, while both GR and MR signaling were required in the regulation of CFTR-II and Na(+), K(+)-ATPase alpha1b. In FW gills, GR1 and MR transcript levels were not significantly affected by cortisol. In SW gills, GR1 and MR transcripts were downregulated by cortisol; GR1 was regulated via the MR and MR regulation was mediated via GR.

Paralytic shellfish toxins in zooplankton, mussels, lobsters and caged Atlantic salmon, Salmo salar, during a bloom of Alexandrium fundyense off Grand Manan Island, in the Bay of Fundy

Substantial mortalities of Atlantic salmon ( Salmo salar) at two aquaculture sites in Long Island Sound, off Grand Manan Island, Bay of Fundy (BoF) (New Brunswick, Canada) in September 2003, were associated with a bloom of Alexandrium fundyense (>3 × 10 5 cells L −1), a dinoflagellate alga that produces toxins which cause paralytic shellfish poisoning (PSP). Cells of A. fundyense collected from surface waters while fish were dying had total paralytic shellfish (PS) toxin concentrations of 70.6 pg STX equiv. (saxitoxin equivalents) cell −1 and PS toxin profiles rich in carbamate toxins (78.2%). The zooplankton sampled contained PS toxins (63.1 pg STX equiv. g −1 wet wt) and the toxin profile matched that of A. fundyense cells. Mean PS toxin levels were low (<4 μg STX equiv. 100 g −1 wet wt) in stomach, gill and muscle tissues of moribund salmon, suggesting that PS toxins are very lethal to salmon. The PS toxin concentrations in blue mussels ( Mytilus edulis) growing on the salmon cages (37; 526 μg STX equiv. 100 g −1 wet wt) were the highest recorded to date from this region. Their PS toxin profiles showed enhanced carbamate contents (85.5%) compared with that found in A. fundyense. Blue mussels collected from an adjacent Canadian Food Inspection Agency (CFIA) monitoring site in Grand Manan had PS toxin concentrations of 4214 and 150 μg STX equiv. 100 g −1 wet wt in late September and December, respectively, well above the regulatory limit (RL), and horse mussels ( Modiolus modiolus) collected in late September had PS toxin concentrations of 2357 μg STX equiv. 100 g −1 wet wt. Detoxification under laboratory conditions suggested that blue mussels may require up to 19 weeks for elimination below RL when they accumulate these high concentrations of PS toxins. This depuration period may be shorter in the field. PS toxin levels above RL were detected in hepatopancreatic tissues of lobster ( Homarus americanus), with lower levels (<16 μg STX equiv. 100 g −1 wet wt) in tail muscle and gills. These results illustrate the movement of PS toxins through the marine food chain following an A. fundyense bloom in the BoF, and support earlier studies suggesting that kills from the region of zooplanktivorous fish, such as herring ( Clupea harengus harengus), can be attributed to blooms of A. fundyense. This is the first reported incident of PSP associated with mortalities of caged Atlantic salmon in the BoF. Analyses of muscle tissues and viscera from the affected salmon indicated that any portion would not be a health hazard if consumed.

Development of an in vivo assay to assess attachment of Neoparamoeba sp. (an amphizoic gymnamoeba) to the gills of Atlantic salmon, Salmo salar L.

Journal of Fish DiseasesVolume 30, Issue 3 p. 187-190 Development of an in vivo assay to assess attachment of Neoparamoeba sp. (an amphizoic gymnamoeba) to the gills of Atlantic salmon, Salmo salar L. P B B Crosbie, P B B Crosbie Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorM B Adams, M B Adams Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorM G Attard, M G Attard Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorB F Nowak, B F Nowak Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this author P B B Crosbie, P B B Crosbie Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorM B Adams, M B Adams Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorM G Attard, M G Attard Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this authorB F Nowak, B F Nowak Aquafin CRC, School of Aquaculture, University of Tasmania, Launceston, Tasmania, AustraliaSearch for more papers by this author First published: 06 March 2007 https://doi.org/10.1111/j.1365-2761.2007.00805.xCitations: 4 P Crosbie, Aquafin CRC, School of Aquaculture, University of Tasmania, Locked Bag 1370, Launceston, 7250 Tasmania, Australia(e-mail: [email protected]) Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume30, Issue3March 2007Pages 187-190 RelatedInformation

Comparison of organotin accumulation in the masu salmon Oncorhynchus masou accompanying migratory histories

In order to examine the accumulation pattern of organotin compounds (OTs) accompanying the migration pattern in diadromous fish, tributyltin (TBT) and triphenyltin (TPT) compounds and their derivatives were determined in the liver, muscle, gill, and ovary tissues of both sea-run and freshwater-resident masu salmon, which are of the same species, Oncorhynchus masou. Their migratory histories were estimated using strontium (Sr) and calcium (Ca) analysis in the otolith. A significant difference in the mean Sr:Ca ratio from the core to the edge of the otolith was found between sea-run and freshwater-resident masu salmon. The TBT concentration in the liver was significantly higher than that in the other tissues in both sea-run and freshwater-resident fishes. In sea-run masu salmon, the TBT concentrations in all tissues except for the ovary were significantly higher than in those of freshwater-resident individuals. In the sea-run type, the percentage of TBT was higher than that of the freshwater-resident type. The TPT concentration in the liver of the sea-run type was also significantly higher than that in the other tissues, while that in the gill of the freshwater-resident type was significantly higher than that in the other tissues except for the ovary. The TPT concentrations found in the liver and muscle of the sea-run type were significantly higher than those in the freshwater-resident type, whereas the values of the gill in the sea-run type were significantly lower than those in the freshwater-resident fish examined. The percentage of TPT in the sea-run type was higher than that of the freshwater-resident type. These results suggest that the sea-run O. masou has a higher ecological risk of TBT and TPT exposure than the freshwater-residents during their life history.

Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

BackgroundThe aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon Salmo salar L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N2) or in RNAlater. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues.ResultsThe results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, Na+-K+-ATPase α1b was significantly downregulated and hypoxia inducible factor 1 (HIF1) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N2 than to use RNAlater.ConclusionProlonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the Na+-K+-ATPase α1b gene and upregulation of the HIF1 gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction.

Hormone receptors in gills of smolting Atlantic salmon, Salmo salar: Expression of growth hormone, prolactin, mineralocorticoid and glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase type 2

This is the first study to report concurrent dynamics in mRNA expression of growth hormone receptor (GHR), prolactin receptor (PRLR), gluco- and mineralocorticoid receptor (GR and MR) and the 11β-hydroxysteroid dehydrogenase type-2 enzyme (11β-HSD2) in Atlantic salmon ( Salmo salar) gill during smoltification. Transcript levels were analysed by quantitative PCR in fresh water (FW) fish and after a 24-h salt water (SW) challenge. GHR transcript levels increased concurrent with gill Na +, K +-ATPase activity in FW fish consistent with the SW-adaptive role of GH. SW-transfer induced an increased GHR expression levels in the early stages of smoltification but a decrease in expression at the peak of smoltification. PRLR transcript levels decreased steadily during smoltification in agreement with the recognized hyper-osmoregulatory role of PRL. Surprisingly, PRLR levels increased after SW transfer during the course of smoltification. GR mRNA levels were low early on during smoltification but increased at the peak of smoltification and remained high during de-smoltification, indicative of increased cortisol signalling at this point. Coherently, SW transfer increased GR levels to smolt levels prior to the smoltification peak. 11β-HSD2 levels increased at the smoltification peak and MR levels increased during de-smoltification, suggesting a need for protection of MR from cortisol signalling during smoltification. This is supported by the fact that SW-transfer results in a profound up-regulation of 11β-HSD2, whereas SW transfer down-regulates MR levels. The study concludes that GR and MR may have distinctive roles in developing hypo- and hyper-osmoregulatory mechanisms during smoltification and de-smoltification, respectively.

Isolation and characterization of acidic glycosphingolipids from the gill of the Pacific Salmon (Oncorhynchus keta): A novel hybrid-type ganglioside with isoglobo- and neolacto-Series

Monosialosyl gangliosides and sulfoglycolipids in the gill of pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. Acidic glycolipid bands (M1-M13) detected by thin layer chromatography were separated by Iatrobeads column chromatography and 13 components were characterized by TLC, compositional analysis, methylation analysis, chemical and enzymatic degradation, liquid secondary ion mass spectrometry and (1)H nuclear magnetic resonance spectroscopy. In addition to the acidic glycolipids with known structures (SM4s, SM3, GM3, LM1, GM1b and V(3)alphaFuc,IV(3)betaGalNAc-GM1a), two fractions (M11 and M13) of unknown monosialosyl gangliosides with TLC mobility slower than GM1a were isolated and characterized as having the following structure with a hybrid of isoglobo- and neolacto-series. [formula: see text] Analysis of fatty acid indicated predominance of C24:1 fatty acid in the upper band (M11) and shorter chain saturated fatty acids in the lower band (M13). The tissue concentrations of M11 and M13 were 1.15 and 0.96 mumol/kg wet weight, respectively.

Structural analysis of a unique hybrid-type ganglioside with isoglobo-, neolacto-, and ganglio-core from the gills of the Pacific salmon ( Oncorhynchus keta)

Monosialosyl gangliosides from the gills of the Pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. The unknown acidic glycolipids (M14 and M15) with slower mobility than GM1a on thin-layer chromatography were separated by Iatrobeads column chromatography and were characterized by compositional analysis, methylation analysis, chemical, and enzymatic degradation, negative-ion LSIMS, and 1H nuclear magnetic resonance spectroscopy. Both M14 and M15 contained a same oligosaccharide core with isoglobo-, neolacto-, and ganglio-series as follows: [Display omitted] The only difference between M14 and M15 was in fatty acid acylation. Analysis of the fatty acids indicated a predominance of C24:1 fatty acid in M14 and shorter chain saturated fatty acids, C14:0 and C16:0, in M15.

Quantitation of immune response gene expression and cellular localisation of interleukin-1β mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, β-actin. Interleukin-1β (IL-1β) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1β mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1β mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1β-specific biotinylated cRNA probe. Expression of IL-1β mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1β at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1β is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1β mRNA expression during infection.

Regiospecificity profiles of storage and membrane lipids from the gill and muscle tissue of atlantic salmon (Salmo salar L.) grown at elevated temperature

Regiospecific and traditional analysis, of both storage and membrane lipids, was performed on gill, white muscle, and red muscle samples taken from Atlantic salmon (Salmo salar) to gauge the effect of elevated water temperature. The fish, fed a commercial diet, were held at an elevated water temperature of 19 degrees C. Total n-3 PUFA, total PUFA, and n-3/n-6 and unsaturated/saturated fatty acid (UFA/SFA) ratios in the FA profile of the total lipid extract in the white muscle were fairly low compared with fish grown at 15 degrees C. Adaptation of structural and storage lipids at elevated temperatures was shown by a significant (P < 0.01) reduction in PUFA especially in the percentage of EPA (6-8%). Further adaptation was indicated by the percentages of SFA, which were significantly (P< 0.05) higher in gill (56%) and white muscle (58%) polar lipid fractions and coincided with lower percentages of n-3, n-6, and total PUFA. The regiospecific profiles indicated a high affinity of DHA to the sn-2 position in both the TAG (61-68%) and polar lipid (35-60%) fractions. The combination of detailed regiospecific and lipid analyses demonstrated adaptation of cell membrane structure in Atlantic salmon grown at an elevated water temperature.

DIFFERENCES IN HOST SELECTION OF ACTINOSPORES OF TWO MYXOSPOREANS, MYXOBOLUS ARCTICUS AND THELOHANELLUS HOVORKAI

We investigated the host selection mechanism of actinospore stages of 2 myxosporeans, Myxobolus arcticus and Thelohanellus hovorkai, infecting masu salmon (Oncorhynchus masou) and common carp (Cyprinus carpio), respectively. Discharge of the polar filaments and sporoplasm release by M. arcticus actinospores occurred within the first 5 min of exposure to skin mucus of masu salmon. The actinospores also reacted to the mucus of nonsusceptible fish, i.e., sockeye salmon (Oncorhynchus nerka) and goldfish (Carassius auratus), although the reactivity was comparatively lower. After exposure of masu, and sockeye and chum salmon (Oncorhynchus keta) to M. arcticus actinospores, the penetration of sporoplasms was observed in the fins and gills of masu and sockeye salmon to a similar extent and to a lesser extent in chum salmon. Thelohanellus hovorkai actinospores exhibited a slow response of sporoplasm release to common carp mucus as well as penetration into the gills of common carp. Neither chemoresponse to mucus of nonsusceptible fish (goldfish and sockeye salmon) nor sporoplasm invasion in goldfish was observed for T. hovorkai actinospores. These results indicate notable differences in the host selection at the time of entry between M. arcticus and T. hovorkai; the former responds quickly to fish mucus with low host specificity, whereas the latter was highly host specific in a dilatory reaction.

Microfauna associated with amoebic gill disease in sea‐farmed Atlantic salmon, Salmo salar L., smolts

A study of microfauna, associated with pathological changes in the gills of Atlantic salmon, Salmo salar L., was conducted over 2001-2002. Monthly samples of 1(+) salmon smolts were taken, protozoan populations were quantified and gill health was assessed histologically. Protozoan densities were correlated with pathological changes, in order to determine their possible role in lesions in the gills. The most severe gill tissue changes were observed in summer/autumn and the least in spring. A diverse polyphyletic protozoan community was observed colonizing the gills, including Neoparamoeba sp., other amoebae, scuticociliates, Ichthyobodo-like flagellates, trichodinid ciliates and prostomatean ciliates. The earlier gill tissue changes in the gill were not always associated with the presence of these microorganisms, whereas amoebae (other than Neoparamoeba sp.), Ichthyobodo-like flagellates and trichodinid ciliates correlated with augmenting gill lesions. Neoparamoeba sp. was present, but its abundance did not correlate with the disease. This study suggests that a diversity of protozoans including Ichthyobodo-like flagellates, trichodinid ciliates and amoebae other than Neoparamoeba sp. are involved in the aetiology of amoebic gill disease in the Irish situation.

Reciprocal expression of gill Na+/K+-ATPaseα-subunit isoforms α1a and α1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance

The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered alpha-subunit isoforms of Na+/K+-ATPase (alpha1a and alpha1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl- and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for the alpha1a isoform quickly decreased following seawater exposure whereas alpha1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the alpha1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms alpha1a and alpha1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.

Interspecific distribution and co-associations of chemical elements in the liver tissue of marine mammals from the Polish Economical Exclusive Zone, Baltic Sea

Concentrations of Al, B, Ba, Cd, Co, Cr, Cu, Fe, Ga, Hg, Li, Mn, Mo, Ni, Pb, Se, Si, Sr, Tl, V, Zn, Ca, K, Mg, Na and P in the livers of marine mammals obtained from by-catches or stranded on beaches on the Polish Baltic coast were determined by ICP-MS or ICP-AES and CV AAS. Interspecific diversity with respect to the contents of these elements was found in cetaceans and pinnipeds. The diverse Cd contents in the livers of these mammals can be attributed to the variable concentrations of this element in their food. Mercury was correlated with age and can reach high concentration associated with higher level of Se in older specimens. No significant relationships were found between concentration of the chemical elements studied and nutritional status/condition of the Baltic harbour porpoises as well as between their concentration in specimens from the Gulf of Gdańsk and open Baltic. It seems that the nutritional and health status of the specimens studied is generally enough good since the specimens studied were not stranded on beach because of starvation but almost incidentally caught in salmon gill nets. Strong correlations were found between the macroelements analysed, i.e. for the Ba–Ca–Sr, Ca–K, Ca–Mg, Mg–P, Zn–Mg and Zn–P assemblies. Significantly higher content of Al was found in males of harbour porpoises.

An EST-based approach for identifying genes expressed in the intestine and gills of pre-smolt Atlantic salmon (Salmo salar)

BackgroundThe Atlantic salmon is an important aquaculture species and a very interesting species biologically, since it spawns in fresh water and develops through several stages before becoming a smolt, the stage at which it migrates to the sea to feed. The dramatic change of habitat requires physiological, morphological and behavioural changes to prepare the salmon for its new environment. These changes are called the parr-smolt transformation or smoltification, and pre-adapt the salmon for survival and growth in the marine environment. The development of hypo-osmotic regulatory ability plays an important part in facilitating the transition from rivers to the sea. The physiological mechanisms behind the developmental changes are largely unknown. An understanding of the transformation process will be vital to the future of the aquaculture industry. A knowledge of which genes are expressed prior to the smoltification process is an important basis for further studies.ResultsIn all, 2974 unique sequences, consisting of 779 contigs and 2195 singlets, were generated for Atlantic salmon from two cDNA libraries constructed from the gills and the intestine, accession numbers [Genbank: CK877169-CK879929, CK884015-CK886537 and CN181112-CN181464]. Nearly 50% of the sequences were assigned putative functions because they showed similarity to known genes, mostly from other species, in one or more of the databases used. The Swiss-Prot database returned significant hits for 1005 sequences. These could be assigned predicted gene products, and 967 were annotated using Gene Ontology (GO) terms for molecular function, biological process and/or cellular component, employing an annotation transfer procedure.ConclusionThis paper describes the construction of two cDNA libraries from pre-smolt Atlantic salmon (Salmo salar) and the subsequent EST sequencing, clustering and assigning of putative function to 1005 genes expressed in the gills and/or intestine.

MHC class II + cells in the gills of Atlantic salmon ( Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon ( Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II + cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II + cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II + cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II + cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.

Morphological presentation of a dendritic-like cell within the gills of chinook salmon infected with Loma salmonae

Morphological presentation of a dendritic-like cell within the gills of chinook salmon infected with Loma salmonae

NOVEL IN VITRO METHOD TO STUDY FISH INTESTINE

Early in life, salmonid fishes move from a freshwater existence to a life in seawater, requiring a complete overhaul of their osmoregulatory systems. The vertebrate intestine is a major osmoregulatory organ and in fish,intestinal salt and water uptake is essential for maintaining water balance. Solute and water movement is mainly driven across the intestinal epithelium by the Na+, K+-ATPase, an ATP-requiring enzyme located on the basolateral membrane or blood side of the intestinal cell. Cortisol, a stress hormone, is thought to mediate the necessary osmoregulatory adaptations required for a salmonid's transformation between environments. It has been suggested that intestinal Na+, K+-ATPase activity, which becomes elevated in preparation for seawater survival, is stimulated by cortisol in many salmonid fishes. However, the influence of cortisol on fish intestine has never been measured directly. To determine the direct effects of cortisol on the intestine during salinity transfer, an in vitrointestinal tissue culture system is needed.Thus, Philip Veillette and Graham Young set out to develop an intestinal tissue culture system from freshwater-adapted sockeye salmon Oncorhynchus nerka by adapting methods used for culturing salmon gill filaments. They decided to culture the pyloric ceca (appendages attached to the salmon's intestines) and the posterior intestine based on their significant roles in absorption and osmoregulation. They cut each intestinal region into pieces,incubated the intestinal preparations at 14°C and gassed them daily with a 95% O2, 5% CO2 mixture to keep the cells alive. To see if their tissue culture was still viable after 3 or 6 days in culture,Veillette and Young examined the preparations using light and transmission electron microscopy. Sure enough, they noticed intact nuclei and organelles,which meant that the intestinal cells were still functional; they had an in vitro intestinal tissue culture.Veillette and Young could now test whether salmon intestinal Na+, K+-ATPase activity is stimulated by cortisol. In the absence of cortisol, the activity of Na+, K+-ATPase in the intestine preparation declined after several days in culture. However,when Veillette and Young treated the preparations with cortisol, they noticed significantly maintained Na+, K+-ATPase activity in both segments of the intestine, demonstrating for the first time that cortisol acts directly on the teleost intestine. The response to cortisol was dose-dependent and specific for cortisol over other corticosteroids within a physiological range. To see if they could replicate these results in vivo, they implanted salmon with slow-release cortisol, elevating plasma cortisol concentrations in these fish. Just as they had found in vitro,cortisol increased Na+, K+ ATPase activity in both intestinal segments in live fish.Using a novel approach, Veillette and Young have shown that the salmon pyloric ceca and the posterior segment of the intestine are direct targets for the osmoregulatory actions of cortisol in salmonids. Their novel culture system could potentially be used to examine the effects of a variety of neuroendocrine factors on the intestine under many different physiological conditions.

Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase

We investigated the relationship between nitric oxide (NO) and Na(+),K(+)-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na(+),K(+)-ATPase alpha-subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA alpha-subunit mRNA revealed cellular colocalization of NKA alpha-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA alpha-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l(-1)) and PAPA-NONOate (NOC-15; 0.5 mmol l(-1)) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na(+),K(+)-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na(+),K(+)-ATPase activity prior to entering seawater.

Effects of dissolved organic carbon and hardness in freshwater used to treat amoebic gill disease

Amoebic gill disease (AGD) is a significant disease of Atlantic salmon farmed in South East Tasmania. The commercial treatment for the disease is a freshwater bath for up to 4 h. Previous studies have shown that the chemical composition of the freshwater, in particular total water hardness, affects the efficacy of the treatment. The aim of this study was to determine if other water chemistry parameters, such as dissolved organic carbon (DOC), interact with total water hardness to affect treatment success. Firstly, the relative survival of isolated gill amoebae incubated for up to 3 h with hard or soft water (346.0 and 34.6 mg L−1 CaCO3 respectively) with low or high concentrations of humic or tannic acid (5 and 50 mg L−1 respectively) was determined. Secondly, fish with AGD were bathed for 2.5 h in hard or soft water (249.3 and 35.3 mg L−1 CaCO3) containing either 5 or 20 mg L−1 humic acid. The number of viable amoebae surviving on the gills and number of gill lesions were determined. It was found that the concentration of DOC used in this study that represents the levels commonly found around SE Tasmania is unlikely to have any commercial significance in the reduction in amoebae on the gills of Atlantic salmon. However, this study provided further support that freshwater selected for bathing AGD-affected salmonids should be chosen primarily on its total water hardness.