Terence M. Bradley

Cloning and characterization of salmon hsp90 cDNA: Upregulation by thermal and hyperosmotic stress

Accumulating evidence suggests that glucocorticoids are essential for development of hypoosmoregulatory capacity in salmon during adaptation to seawater. Heat shock protein (hsp)90 has been reported to function in signal transduction and the maturation and affinity of glucocorticoid receptors. We sought to determine whether this hsp might be upregulated by thermal and hyperosmotic stress in salmon, a species that migrates between the freshwater and marine environments. A 2625-bp cDNA cloned from a salmon cDNA library was found to code for a protein of 722 amino acids exhibiting a high degree of identity with zebra fish (92%) and human (89%) hsp90beta. Accumulation of hsp90 mRNA was observed in isolated branchial lamellae incubated under hyperosmotic conditions and in branchial lamellae of salmon exposed to hyperosmotic stress in vivo. In contrast, exposure of kidney to hyperosmotic stress in vitro and in vivo failed to elicit an increase in the quantity of hsp90 mRNA. By way of comparison, accumulation of hsp90 mRNA was observed in both branchial lamellae and kidney tissue subjected to thermal stress in vitro and in vivo. Western blot analyses of proteins isolated from tissues under identical conditions in vitro revealed that the pool of hsp90 increased with thermal stress but not with osmotic stress. The results suggest that accumulation of hsp90 mRNA in response to osmotic stress is unrelated to cellular protein denaturation and that synthesis of hsp90 may be regulated at both the level of transcription and translation.

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.

Thermal shock of salmon in vivo induces the heat shock protein hsp 70 and confers protection against osmotic shock

Abstract Salmon transferred from freshwater hatcheries to seawater netpens are subjected to osmotic shock which can disrupt biochemical processes and cause stunted growth or death. The present study aimed to determine if heat shock proteins (hsps) induced by exposure of living animals to thermal shock might confer protection against this osmotic challenge. A 66 kDa protein which cross reacted with a monoclonal antibody to mammalian hsp 70, was induced in hepatic and branchial tissue of juvenile salmon subjected to a 15 min heat shock at 26°. De novo synthesis of this protein did not continue beyond the first 3 h of recovery from heat shock, but the newly synthesized protein was stable for at least 12 h. Heat shocked salmon were better able to survive a subsequent severe osmotic challenge (45 ppt). Cross protection against osmotic shock was observed only during a two month period coincident with parr–smolt transformation, when branchial Na + /K + ATPase activity was elevated. This report is the first to demonstrate protection against osmotic challenge by heat shock in a living animal.

Heat shock proteins are not sensitive indicators of hatchery stress in salmon

Abstract An array of physiological, endocrinological, biochemical and behavioral indicators have been investigated for utility in assessing the level of stress imposed on cultured finfish by rearing conditions and husbandry practices. In the present report, juvenile Atlantic salmon (Salmo salar) were exposed to common forms of hatchery stress and the response of heat shock proteins (hsp) 30, 70 and 90 were measured as possible indicators of stress. Treatments included exposure to two types of anesthesia (tricaine methanesulfonate and 2-phenoxyethanol), formalin, hypoxia, hyperoxia, capture stress, crowding, feed deprivation and cold stress. Exposure of fish to heat stress at 26 °C (ΔT=11 °C) served as a positive control, and untreated fish were used as a negative control. Total RNA was isolated from gill tissue following treatment, and subjected to Northern analysis with cDNA probes specific to the three hsps. Hsp70 and Hsp90 mRNA levels increased three-fold and two-fold above control levels, respectively, following 15-min heat stress. hsp30 mRNA levels were unaffected by 15-min exposure to heat stress, but increased two-fold over control levels following 30-min exposure. In contrast, Hsp mRNA was not upregulated in response to the different hatchery stresses examined. Although cold stress, crowding and capture stress caused an increase in hsp90 mRNA levels, these were not significant. The findings suggest that hsp30, hsp70 and hsp90 are not sensitive indicators of hatchery stress in Atlantic salmon.

The effects of photoperiod manipulation on the reproduction of black sea bass, Centropristis striata

The black sea bass, Centropristis striata, is a potential candidate for commercial aquaculture in the United States. One of the most significant constraints limiting commercial production of black sea bass is the lack of a reliable supply of eggs and larvae. A narrow window of annual spawning prevents year-round availability of juveniles for grow out. The present study reports on the use of photoperiod manipulation to alter the timing of spawning in C. striata and increase availability of larvae. Adult black sea bass were maintained under a simulated natural (SNP) or an accelerated long day (ALD) photoperiod regimen. Water temperature was adjusted from 14 to 20 °C to correspond to seasonal changes in daylength. Reproductive development was monitored by histological analysis of gonadal biopsies and plasma levels of sex steroids (estradiol-17β, testosterone and 11-ketotestosterone). Females in both regimens were induced to spawn by implantation of 25 μg luteinizing hormone-releasing hormone analogue (LHRHa) when follicle diameters exceeded 500 μm. Acceleration of the photoperiod advanced the annual cycle of circulating gonadal steroids in both sexes without altering the profile. The onset of spermiation in males was unaffected by photoperiod manipulation, but follicle growth and spawning in females were advanced approximately 2 months by exposure to the ALD regimen. Of interest, the ALD photoperiod regimen accelerated the time frame of sexual succession from female to male without reducing the mean volume of eggs produced per female. These findings suggest that photoperiod manipulation can be used to alter the timing of reproduction in black sea bass and increase the supply of eggs for commercial aquaculture.

Liver ultrastructure of juvenile Atlantic salmon (Salmo salar)

Light and transmission electron microscopy of the liver of juvenile Atlantic salmon (Salmo salar) reveals a tubular arrangement of parenchymal cells, with biliary passages typically located at the center of tubules. Hepatocytes generally contain a single nucleus surrounded by a cuff of rough endoplasmic reticulum (RER), with many round to elongate mitochondria associated with the perinuclear RER. Whereas glycogen deposits are common and usually lie at the cell periphery, parenchymal cells seldom contain lipid droplets. Golgi complexes and heterogeneous dense bodies also occur in many hepatocytes, often in close proximity to bile canaliculi. Numerous microvilli from hepatocytes extend into the subendothelial space of Disse, which is also the location of stellate fat‐storing cells. Interhepatocytic macrophages, sometimes containing prominent phagolysosomes and residual bodies, are common in the liver. The intrahepatic biliary system consists of intercellular canaliculi, bile pre‐ductules, ductules, and ducts. In contrast to some other teleosts, the liver of the Atlantic salmon contains no intracellular bile canaliculi or Kupffer cells. The hepatic endothelium, arterioles, and perivenous regions are also described.

l-Alanine binding sites and Na+, K+-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.

High-affinity Ca2+,Mg2+-ATPase in plasma membrane-rich preparations from olfactory epithelium of Atlantic salmon

High-affinity Ca2+,Mg(2+)-ATPase was identified in a plasma membrane-rich fraction of olfactory epithelium from Atlantic salmon (Salmo salar). The enzyme required both Ca2+ and Mg2+ for activation. The apparent Km for Ca2+ was 9.5 nM and Vmax was 0.85 mumol Pi/mg of protein per min. Stimulation by Ca2+ was optimal at 5-100 microM MgCl2. Bovine brain calmodulin had no effect on Ca2+,Mg(2+)-ATPase, even after multiple washes of the membrane preparation with EDTA or EGTA. Endogenous calmodulin was somewhat resistant to removal and could be detected with immunoblotting after multiple washes of the membrane preparation with EDTA or EGTA. This endogenous calmodulin may regulate Ca2+,Mg(2+)-ATPase activity because the activity was inhibited by calmidazolium. Vanadate inhibited Ca2+,Mg(2)-ATPase activity and thapsigargin, a specific inhibitor for Ca2+,Mg(2+)-ATPase of endoplasmic reticulum, had no effect on the enzyme activity. High affinity Ca2+,Mg(2+)-ATPase exists in both ciliary and nonciliary membranes with a similar Km for Ca2+. Ca2+,Mg(2+)-ATPase activity is greater in cilia preparations than in membranes from the deciliated olfactory epithelium. As a putative plasma membrane Ca2+ pump, this high-affinity Ca2+,Mg(2+)-ATPase may play an important role in the regulation of intracellular Ca2+ in olfactory epithelia. In particular, the ciliary membrane may play a prominent role in the removal of Ca2+ from ciliated olfactory receptor cells after odorant stimulation.

L-carnitine protects fish against acute ammonia toxicity.

1. Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally (i.p.) with 0.25 M mannitol followed 1 hr later by an i.p. challenge of ammonium acetate. 2. At 10.75 mmol ammonium acetate/kg body weight, 98% of the fish showed signs of ammonium toxicity and 69% died. 3. Substitution of L-carnitine (10-16 mmol/kg) for mannitol afforded striking protection from the subsequent challenge with ammonium acetate; 67% showed no signs of ammonia toxicity and only 4% died. 4. Of other quaternary amines tested, trimethylamine oxide also afforded protection, but betaine and choline did not.

Comparative microbial diversity in the gastrointestinal tracts of food animal species

Abstract Molecular tools based on small subunit (SSU) rDNA gene sequences offer a powerful and rapid tool for the analysis of complex microbial communities found in the gastrointestinal tracts (GIT) of food animal species. Extensive comparative sequence analysis of SSU rRNA molecules representing a wide diversity of organisms shows that different regions of the molecule vary in sequence conservation. Oligonucleotides complementing regions of universally conversed SSU rRNA sequences are used as universal probes, while those complementing more variable regions of sequence are useful as selective probes targeting species, genus, or phylogenetic groups. Different approaches derive different information and this is highly dependent on the type of target nucleic acid employed and the conceptual and technical basis used for nucleic acid probe design. Generally these approaches can be divided into DNA-based methods employing empirically characterized probes and rRNA-based methods based on comparative sequence analysis for design and interpretation of “rational” probes. Polymerase chain reaction (PCR) based techniques can also be applied to the analysis of microbial communities in the GIT. Direct cloning of SSU rDNA genes amplified from these complex communities can be used to determine the extent of diversity in these GIT communities. Denaturing gradient gel electrophoresis (DGGE) is another powerful tool for profiling microbial diversity of microbial communities in GI tracts. Sequence analysis of the excised DGGE amplicons can then be used to presumptively identify predominant bacterial species. Examples of how these molecular approaches are being used to study the microbial diversity of communities from steers fed different diets, swine fed probiotics, and Atlantic salmon fed aquaculture diets are presented.