Heat Shock Proteins in Branchial Tissue in Atlantic Salmon (Salmo salar)

Lamellae isolated from gill arches of Atlantic salmon were incubated in 15, 25 and 21°c. Five heat shock or stress proteins (hsps) with molecular weights of 54, 71, 72, 82 and 87 kDal appeared after incubation at 2s c. At 21°c all five proteins were induced in greater quantity and an additional protein of 67 kDal was observed. The time 0 required for the induction of hsps at 25 C was determined by labeling the gills in vitro for one hour after intervals of up to four hours of heat shock. All five proteins were apparent after one hour of heat shock and maximal by two hours. The lamallae continued to synthesize hsps throughout the four hours. Stress proteins were not induced in lamallae exposed to 25 to 300 uM of sodium arsenite or 50 to 500 mM sodium chloride. Although viability was high under these conditions, overall protein synthesis was suppressed. Lamellae proteins induced by heat shock at 25°c were incubated with monoclonal and polyclonal antibodies to hsps 60, 70 and 90. Only the antibodies to constitutive/inducible hsp 70 and the polyclonal antibody to hsp 70 exhibited different degrees of binding in control and shocked samples. October and January fish were subjected to osmotic shock in vivo by transfer from freshwater to 27 ppt and 35

all five proteins were induced in greater quantity and an additional protein of 67 kDal was observed. The time 0 required for the induction of hsps at 25 C was determined by labeling the gills in vitro for one hour after intervals of up to four hours of heat shock. All five proteins were apparent after one hour of heat shock and maximal by two hours. The lamallae continued to synthesize hsps throughout the four hours.
Stress proteins were not induced in lamallae exposed to 25 to 300 uM of sodium arsenite or 50 to 500 mM sodium chloride. Although viability was high under these conditions, overall protein synthesis was suppressed. increased to meet the growing human ~onsumption of salmon.
In 1983, the U.S. imported approximately 3,968 metric tons of Atlantic salmon. Six years later, imports increased nearly 4.5 times to more than 17,000 metric tons (Bettencourt -and Anderson, 1990). The increased demand and higher prices could allow domestic aquaculturists to profitably produce and market salmon.
In commercial salmon aquaculture fish are typically reared in seawater netpens because they cannot be produced cost effectively in terrestrial-based facilities. The fish are directly transferred from freshwater tanks to seawater vi net pens. The fish are reared in these units at relatively low cost to market size in about 18 months. Salmon can only be transferred to seawater during a short period of time called the parr-smolt transformation or smoltif ication (Hoar 1988(Hoar , 1976. Many morphological and physiological changes occur during smoltification and enable the fish to successfully make the transition from a hydrating (freshwater) to a dehydrating (seawater) environment.
Smoltif ication occurs in the spring during the increase in day light and water temperature. When fish are transferred outside the two to four week period of smoltif ication, there are high mortalities and poor growth of survivors (Hoar, 1976).
In restoration programs non-smelting salmon released into the rivers remain in the stream where they are exposed to predators and food availability is low. These fish are not likely to survive and return to the river to spawn.
Smoltification must be identi~ied accurately for commercial aquaculture and restoration programs.   Fluorograph depicting proteins from lamallae exposed to 25°c and labeled for two hours then incubated without radiolabel for various times at 25°c. 52 Figure 9. Protein concentrations of lamallae exposed to 25°c and labeled for two hours then chased for various times at 15°c. 54 Figure 10. Fluorograph depicting proteins from lamallae exposed to 25°c and labeled for two hours then incubated without radiolabel for various times at 15°c.     deviation in fish exposed to in vivo osmotic shock (IV2). 88 Figure 27. Incorporation of radiolabel into protein fish exposed to in vivo osmotic shock (IV2). 90 Figure 28. Fluorograph depicting branchial proteins from fish subjected to various salinities for two hours.

INTRODUCTION
When an organism is subjected to elevated temperatures, a group of polypeptides called heat shock proteins (hsps) is induced. These proteins are thought to protect the organism from further heat shock. Other environmental stresses, such as exposure to sodium arsenite, heavy metals, ethanol and hypoxia also induce the synthesis of hsps and additional stress proteins (Lindquist 1986, Lindquist andCraig 1988).
Many species of plants (Czarnecka et al, 1988), animals (Welch and Suhan, 1986) and bacteria (Perisic 1989;Taura et al. 1989;Skowyra et al. 1990) have been examined and all have been shown to produce heat shock proteins (for review see Nover, 1991;Sanders, 1993 (Lindquist, 1986). The high degree of homology suggests a low degree of tolerance for mutation and a vital role in sustaining life.
A large variety of stresses induce heat shock proteins.
Members of the hsp 70 family are induced in most organisms by anoxia and nutrient starvation (Nover, 1991). A multitude of chemicals, such as hydrogen peroxide, arsenite and ethanol also induce hsps. However, species-specific responses and threshold levels of induction have been observed. Several organisms synthesize stress proteins in response to various stresses that are not induced by heat shock. These proteins and those induced upon heat shock are broadly termed stress proteins.
The hsps synthesized by these organisms fall into five categories based on molecular size. The family with the greatest molecular weight is the hsps 90. This group of polypeptides is abundant during non-stressed conditions, but during exposure to sublethal or lethal temperatures there is an increase in synthesis of these proteins (Collier and Schlesinger, 1986). These constitutive proteins are located primarily in the cytoplasm with a slightly larger member (hsp 94; also called grp 94) found in the endoplasmic reticulum (Sorger and Pelham, 1987) . . Hsp 94 is induced during glucose starvation, exposure to heat, steroids and other agents. The cytosolic members of hsp 90 have been found to associate with steroid hormone receptors (Dalman et al. 1989;Ohara-Nemoto et al. 1990). Hsp 90 prevents the receptors from binding to DNA when the hormone is not attached and enhances the affinity o~ the receptor for the hormone.
The most abundant and genetically conserved group of heat shock proteins is the hsp 70 family. This group has been extensively studied. Hsp 70 has both constitutive and inducible members with various functions.
Hsps 72 and 73 also interact with maturing polypeptides, folding the polypeptide into the active form (Beckman et al, 1990). Hsp 70 also acts to chaperone the newly synthesized proteins to the appropriate cell destination by preventing improper binding to other proteins (Beckmann et al, 1990;Ellis, 1987). Researchers have hypothesized that hsp 70 also acts to signal the cell of temperature changes (Craig and Gross, 1991).
Hsp 60 has only recently been observed and its functions have not been fully determined. Hsp 60 is constitutive and is synthesized in the cytosol then translocated into the mitochondria. Researchers have shown that hsp 60 prevents the inactivation of dihydrofolate reductase during heat shock (Martin et al, 1992).
The small hsps have molecular weights ranging from 16 to 40 kDal. This family is very div~rse and not highly conserved among species · (Mansfield and Key, 1987). Although the small hsps have not been well studied, some members have been found to be induced during different stages of development without exposing the organism to stress (Cheney and Shearn, 1983). Several investigators suggest they may be important to ontogeny.
The smallest group of heat shock proteins are the ubiquitins (Bond andSchlesinger 1985, Collier andSchlesinger 1986). These are small polypeptides (about 76 amino acids) found in all eukaryotic cells examined (Matthews and VanHolde, 1990 (Craig and Jacobsen, 1984). During embryonic development of many organisms, hsps are not yet synthesized and the organisms are very sensitive to thermal killing. When the organism is capable of synthesizing hsps, it will tolerate temperatures that would be lethal at the embryonic stage. These findings support the hypothesis that stress proteins protect cells (Brown et al, 1992).
Generally the upper lethal limit is the temperature at which vital organs fail (Prosser and Nelson, 1981). One potential mechanism explaining this is the destruction of cellular membranes as they become fluid and loose structure.
The loss of membrane integrity impairs membrane permeability and interferes with the stability of membrane enzymes (Bowler, 1981). Heat labile proteins are denatured at elevated temperatures and may bind non-specifically, causing formation of large aggregates. The cell also responds to heat shock by decreasing synthesis of non-hsp polypeptides.
As ectotherms, physiological processes of fish are greatly affected by temperature fluctuations. Heat shock proteins may be of particular significance in ectotherms because of the wide fluctuations in body temperature that parallel environmental changes. At high temperatures fish react with hyperexcitability, uncoordinated swimming, loss of equilibrium and muscle spasms (Prosser and Nelson, 1981).
Hsps have been found in all eight species of fish examined to date. Tail fin of meda~a (Oryzias latipes) synthesizes three major · heat shock proteins at elevated temperatures in vitro (Oda et al, 1991). Gill, muscle and brain tissues have been shown to synthesize hsps in fathead minnow (Pimebhales promelas) (Dyer et al, 1991(Dyer et al, , 1993 and goldfish (Carassius auratus) tail fin cells synthesize four groups of hsps in vitro (Sato et al, 1990). Rainbow trout hepatoma (Oncorhynchus mykiss) and chinook salmon embryonic cell (Oncorhynchus tshawytscha) lines also produce hsps in response to temperature shock and heavy metals (Kothary et al. 1984;Misra 1989). Winter flounder (Pleuronectes ameri6anus) renal proximal-tubule cells p r oduce hsps from three families at mild and severe heat shock (Brown et al., 1992) Previously, researchers had not determined if Atlantic salmon respond in a similar manner. The natural range of Atlantic salmon is limited to the north Atlantic Ocean.
However, they are cultured in various areas of the world and often encounter stressful thermal conditions in both native and hatchery environments. Although, Atlantic salmon are 0 tolerant of temperatures ranging from freezing to 26 C, they 0 grow better at temperatures below 16 c (Piper et al, 1982) and are frequently exposed to elevated water temperatures, especially during migration. It seems probable that synthesis of hsps might serve as a defense mechanism in many species of ectotherms, including Atlantic salmon, during exposure of close to sublethal temperatures.
Salmon are also exposed to another significant environmental stress, osmotic shock. Juveniles migrate from freshwater to seawater during smoltification and adults return from seawater to freshwater to spawn. In culture situations, juvenile salmon are moved abruptly from freshwater to seawater without the benefit of gradual acclimation. Salmon transferred to seawater experience a transient increase in blood sodium concentration from 175 mM/l in freshwater to ~ 200 mM/l in seawater after 18 hours.
To survive this rise in environmental osmolarity, salmon must regulate the influx of sodium and maintain circulating levels at < 200 mM in the face of environmental sodium levels> 550 mM (Stagg et al, 1989). Functional smelts with hypoosmoregualtory ability are able to reduce sodium and chloride concentrations, while sodium levels in parr remain elevated (Birt et al., 1990). It seems likely that a mechanism is present to prevent cellular damage and death Gill arches were excised with scissors and cut just above the septa with methanol-cleaned razor blades to separate the lamellae (McCormick and Bern, 1989

ANTIGEN SPECIFICITY
Atlantic salmon hsps were tested for antigenic homology to known hsps from other species using Western blotting (Towbin et al, 1979;Dalman et al, 1989). Branchial proteins from control and shocked fish were separated on a gradient gel as described previously and transferred to Immobilon-P membranes (Millipore, Bedford, MA) using a semi-dry electrotransfer system (American Bionetics PolyBlot, Hayward, CA). Transferred proteins were exposed to several antibodies: . mouse monoclonal antibody specific Only primary antibodies that produced bands were tested for specificity. Specificity of antibody binding and silver enhancement was determined by cutting and removing the region of the strip containing the antigen and exposing the remaining proteins to primary and secondary antibodies and silver enhancement. Non-specific binding of the secondary antibody was determined by exposing the strip to secondary antibody and -silver enhancement. Specificity of the silver enhancement treatment to the secondary antibody was tested by exposing the strip to the primary antibody and enhancement. Non-specific binding of silver to the membrane or proteins was determined by excluding primary and secondary antibodies from the procedure.

IN VIVO OSMOTIC SHOCK
To determine if Atlantic salmon produce stress proteins in response to salt shock in vivo, two year old fish were radiolabeled and exposed to low and high salt concentrations in aquaria supplied with aerated, freshwater or seawater ( increasing to approximately 5% in six hours. When the temperature was raised to 21°c, few cells died by four hours of exposure. Cell death increased rapidly to about 15% by the next sampling time at six hours. At 2s 0 c, cell death increased rapidly to 17% within four hours. When the temperature was raised to 3o 0 c or higher, the cells began to die rapidly with 10% mortality within the first hour and 22% by two hours. In preliminary investigations, branchial tissue displayed a very low rate of protein synthesis (Figure 2).
Less than 0.5% of the radiolabeled methionine that crossed into the cells was incorporated into branchial proteins, whereas greater than 30% of the radiolabel was incorporated into hepatic proteins (Wang, J., unp~blished data). The Sodium arsenite did not induce hsps in Atlantic salmon gill tissue. Kothary and Candido (1982) found sodium arsenite effectively induced hsps in a rainbow trout cell line when the cells were allowed a recovery time. Five hsps were induced upon exposure to increased temperatures with molecular weights of 30, 32, 42, 70 and 87 kDal (Kothary and candido, 1982). Upon exposure to 50 uM of sodium arsenite, the same hsps were induced with the addition of a 62 kDal polypeptide. With no recovery time, very few and faint bands were detected and normal protein synthesis was reduced. When fathead minnows were exposed to sodium arsenite various tissue specific proteins were synthesized (Dyer et al., 1993). Gill tissue synthesized five stress proteins with molecular weights of 20, 40, 70, 72 and 74 kDal in response to arsenite. Hsps were evident by two hours, but did not reach maximal synthesis until six to 10 hours of exposure. Four hours may not be long enough for stress proteins to be detected in Atlantic salmon.
Protein synthesis during recovery time differs from protein synthesis during exposure to the stress (Welch and Suhan, 1985). Kothary and Candido (1982) and Dyer et al. (1991)  This could be due to technical problems with the antibody.
It could also show that the lower molecular weight protein of Atlantic salmon has limited homology to hsp 72 found in humans.
Polyclonal antibodies to mammalian hsp 60, did exhibit slight binding to Atlantic salmon polypeptides in the 60 kDal molecular weight range when blocking buff er was not used. These bands were .not detected by any other antibody.
However, they did not appear to change between control and heat shocked groups.
In radiolabeling experiments, a polypeptide band was detected with a molecular weight of 54 kDal. This band was relatively faint and may not have been present in sufficient quantity to enable visualization of differences between control and shocked tissues.
Alternatively, hsp 54 from Atlantic salmon may not share antigenic similarities with human hsp 60. When fish from IV2 were subjected to osmotic shock, no inducible proteins were observed. Plasma chloride levels from fish exposed to 27 ppt for 4 to 48 hours rose above control levels, but did not reach the levels of fish shocked with 27 ppt from IVl. Time of year, photoperiod and stage of development of fish may account for differences in the two in vivo experiments. Typically, smoltification occurs in late April for fish reared at our facility. However, numerous investigations suggest that many of the changes associated with the parr-smolt transformation may begin to appear many months prior to smoltification (Wagner, 1974, Saunders andHenderson, 1978). Constant light photoperiod (L24) is believed to suppress smoltification, however, it may not inhibit smoltification completely (Saunders et al, 1985). Figure 1. Viability of branchial tissue exposed to heat shock determined by lactate dehydrogenase leakage.          Incorporation of radiolabel into protein fish exposed to in vivo osmotic shock (IV2). Values are the mean ± SD of three to five individuals and were analyzed for differences in means using a within groups analysis of variance and a Tukey post-hoc test (alpha = 0.05). Same letter denotes no statistical differences between group means.