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High sensitivity of skin cathepsins L and B of European eel (Anguilla anguilla) to thermal stress
Aquaculture 182 Ž2000. 209–213 www.elsevier.nlrlocateraqua-online
High sensitivity of skin cathepsins L and B of European eel žAnguilla anguilla / to thermal stress Futoshi Aranishi
)
DiÕision of Physiology and Molecular Biology, National Research Institute of Fisheries Science, Yokohama 236-8648, Japan Accepted 4 July 1999
Abstract The changes in bacteriolytic and proteolytic activities of skin cathepsins L and B were studied in thermal-stressed European eel Ž Anguilla anguilla.. When fish were kept in 108C water, all activities increased to reach maximum levels at Day 8 with varying increasing rates of 3.0 Žproteolysis., 4.2 Žlysis of FlaÕobacterium columnare., 5.7 Žlysis of Vibrio anguillarum. and 7.8 Žlysis of Edwardsiella tarda. fold. High water temperature at 308C also induced marked increases in these activities, and the increasing rates were evaluated as 1.5 Žbacteriolysis. –2.0 Žproteolysis. fold higher than those at 108C. Results show a high sensitivity of eel skin cathepsins in bacteriolysis and proteolysis to thermal stress. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Thermal stress; Cathepsins L and B; Bacteriolysis; Skin; Anguilla anguilla
1. Introduction The aquatic environment is rich in pathogenic bacteria that often produce severe disease in fish ŽAustin and Austin, 1993.. Bacterial fish disease is a global problem in fish farming industry, because it is of major significance when fish are under stressful conditions in aquaculture ŽIwama et al., 1997.. Although bacterial fish disease occurs over a wide range of water temperature, bacteriosis is generally enhanced with rising temperature ŽThune et al., 1993.. The thermal effect on fish antibacterial defense therefore needs to be clarified in aquaculture ŽBly et al., 1997.. Anguilliformes, that are extensively farmed species in Asia and Europe, retain novel antibacterial proteases in the skin ŽAranishi and Nakane, 1997a,b.. These proteases, )
Tel.: q81-45-7887645; fax: q81-45-7885001; E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 7 5 - 6
210
F. Aranishir Aquaculture 182 (2000) 209–213
identified as cathepsins L and B, are secreted by skin epidermal cells ŽAranishi et al., 1998a. and catalyze lysis of bacteria ŽAranishi, 1999a,b.. Their activities significantly increase during bacteriosis via the skin ŽAranishi et al., 1998b., but the thermal effects on them remain unclear. The present study verifies bacteriolytic and proteolytic activities of skin cathepsins L and B in thermal-stressed European eel, Anguilla anguilla.
2. Materials and methods 2.1. Experimental procedure Adult European eel, reared to a mean weight of 180 g, were obtained from a commercial fish farm and kept in a 60 l well-aerated and filtered laboratory aquarium at 20.0 " 0.58C over 60 days to allow for acclimation and evaluation of fish health. The experiment on thermal stress employed two groups of five fish that were kept separately at 10.0 " 0.88C and 30.0 " 1.08C for up to 10 days ŽAranishi et al., 1998b.. During the experimental period, the dorsal skin was sampled from a fish at an area of 2 cm2 without overlap every other day and used in prepararing the skin extract ŽAranishi and Nakane, 1997b.. Protein concentration of the extract was measured by a DC Protein Microassay ŽBio-Rad.. 2.2. Bacteriolytic assay Aliquots of the skin extract were incubated with formalin-killed FlaÕobacterium columnare, Vibrio anguillarum or Edwardsiella tarda in 10 mM sodium acetate buffer, pH 5.0, at 378C for 10 min then 208C for 5 min ŽAranishi, 1999a.. The turbidity of the assay mixture was measured at 550 nm before and after reaction Ž1 unit s 0.01 turbidity decreasermin.. 2.3. Proteolytic assay Aliquots of the skin extract were incubated with 2 mM carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide ŽPeptide Institute. in 80 mM sodium acetate buffer, pH 5.0, at 378C for 10 min ŽAranishi and Nakane, 1997a.. The fluorescence of 7-amino-4-methylcoumarin ŽAMC. was measured at an excitation of 380 nm and emission of 460 nm Ž1 unit s 1 nmol AMC liberationrmin..
3. Results 3.1. Low water temperature Fig. 1 shows increases in the activities to lyse three species of pathogenic bacteria in the skin of European eel kept at 108C. The increase in the lysis of E. tarda proceeded
F. Aranishir Aquaculture 182 (2000) 209–213
211
Fig. 1. Skin catheptic bacteriolysis of F. columnare ŽA., V. anguillarum ŽB. or E. tarda ŽC. and proteolysis ŽD. in European eel at 108C Žmean"S.D., ns 5..
linearly up to Day 4, and the lysis of F. columnare or V. anguillarum increased at a high or low rate, respectively. These activities reached similar levels at Day 6, and their maximum increases commonly occurred from Day 6 to Day 8. The increasing rates from Day 2 to Day 8 were evaluated to be F. columnare- V. anguillarum- E. tarda between 4.2 and 7.8-fold. The decreases in the lysis of E. tarda and V. anguillarum after Day 8 were larger than the decrease in the lysis of F. columnare. As well as the bacteriolytic activities, the proteolytic activity increased from Day 2 to Day 8 and then decreased. Its increasing rate from Day 2 to Day 8 was evaluated to be 3.0-fold and less than the increasing rates of the bacteriolytic activities. 3.2. High water temperature The changing profiles of the bacteriolytic activities in the eel skin kept at 308C resembled those at 108C and consequently showed a high sensitivity of skin cathepsins to thermal stress. All of the bacteriolytic activities also reached maximum levels at Day 8, and the order of the increasing rates was the same as that at 108C ŽFig. 2.. Their maximum levels were, however, estimated as 1.5-fold higher than those at 108C. The
212
F. Aranishir Aquaculture 182 (2000) 209–213
Fig. 2. Skin catheptic bacteriolysis of F. columnare ŽA., V. anguillarum ŽB. or E. tarda ŽC. and proteolysis ŽD. in European eel at 308C Žmean"S.D., ns 5..
thermal stress of high water temperature therefore stimulated skin catheptic bacteriolysis more intensely than that of low water temperature. This appeared to be similar for the proteolytic activity, and the increasing rate from Day 2 to Day 8 was equivalent to 2.0-fold higher than at 108C.
4. Discussion This study first demonstrates the high sensitivity of eel skin cathepsins L and B in bacteriolysis and proteolysis to thermal stress. It suggests that their host defense ability is accelerated under stressful conditions, in which bacteriosis frequently occurs. An array of host defense substances in fish skin are either individually or cooperatively stimulated to respond to a variety of environmental stress, such as temperature, osmosis, pollution and density, in aquaculture facilities ŽRice et al., 1996; Bly et al., 1997; Iwama et al., 1997.. In particular, thermal stress is of great significance in bacterial infection, because bacterial disease occurs over a wide range of water temperature ŽThune et al., 1993..
F. Aranishir Aquaculture 182 (2000) 209–213
213
European eel is usually reared around 208C in Japanese aquaculture facilities. In parallel with the thermal-stressed experiments presented in this paper, no significant alteration was observed in various defense activities in the eel skin including both bacteriolytic and proteolytic activities due to cathepsins L and B, when fish were kept at 20.0 " 0.58C under the laboratory conditions Žnot shown.. However, Figs. 1 and 2 show marked increases in these catheptic activities within a few days at both 108C and 308C. These results indicate that an activation mechanism of eel skin cathepsins, probably depending on maturation from their precursory forms, is very susceptible to thermal stress. It is likely that the enhancement of potential catheptic activities in fish skin to respond to thermal stress could contribute to that of resisting ability of fish to bacterial infection. Three bacterial species employed as substrates for eel skin cathepsins are all potential pathogens with extensive fresh-water fish including Anguilliformes ŽAustin and Austin, 1993.. It is known that the bacterial fish pathogens of major importance are, with few exceptions, gram-negative, which have the membrane on the outside of the proteoglycan wall. Aranishi Ž1999a. reports that skin cathepsins L and B of Japanese eel, Anguilla japonica, catalyze lysis of V. anguillarum in an acidic region, and their catalytic action depends on the degradation of specific proteins of the bacterial outer membrane ŽAranishi, 1998.. In this study, the lysis of F. columnare and E. tarda in addition to V. anguillarum is likely to be caused by the same proteolytic action of skin cathepsins of European eel on the bacterial outer membrane, because of the synchronized changes of proteolytic with bacteriolytic activities.
References Aranishi, F., 1998. Cathepsins B and L responsible for defense bacteriolysis in Anguilliform skin. Proc. Int. Symp. Aquat. Anim. Health 3, 222. Aranishi, F., 1999a. Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel. Fish Physiol. Biochem. 20, 37–41. Aranishi, F., 1999b. Possible role for cathepsins B and L in bacteriolysis by Japanese eel skin. Fish Shellfish Immunol. 9, 61–64. Aranishi, F., Nakane, M., 1997a. Epidermal proteases of the Japanese eel. Fish Physiol. Biochem. 16, 471–478. Aranishi, F., Nakane, M., 1997b. Epidermal proteinases in European eel. Physiol. Zool. 70, 563–570. Aranishi, F., Mano, N., Hirose, H., 1998a. Fluorescence localization of epidermal cathepsins L and B in the Japanese eel. Fish Physiol. Biochem. 19, 205–209. Aranishi, F., Mano, N., Nakane, M., Hirose, H., 1998b. Epidermal response of the Japanese eel to environmental stress. Fish Physiol. Biochem. 19, 197–203. Austin, B., Austin, D.A., 1993. Diseases in farmed and wild fish. In: Laird, L.M. ŽEd.., Bacterial Fish Pathogens. Ellis Horwood, Chichester, pp. 263–296. Bly, J.E., Quiniou, S.M., Clem, L.W., 1997. Environmental effects on fish immune mechanisms. Dev. Biol. Stand. 90, 33–43. Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B., 1997. Fish Stress and Health in Aquaculture. Cambridge Univ. Press, Cambridge. Rice, C.D., Kergosien, D.H., Adams, S.M., 1996. Innate immune function as a bioindicator of pollution stress in fish. Ecotoxicol. Environ. Saf. 33, 186–192. Thune, R.L., Stanley, L.A., Cooper, K., 1993. Pathogenesis of gram-negative bacterial infections in warm water fish. Annu. Rev. Fish Dis. 3, 37–68.
High sensitivity of skin cathepsins L and B of European eel žAnguilla anguilla / to thermal stress Futoshi Aranishi
)
DiÕision of Physiology and Molecular Biology, National Research Institute of Fisheries Science, Yokohama 236-8648, Japan Accepted 4 July 1999
Abstract The changes in bacteriolytic and proteolytic activities of skin cathepsins L and B were studied in thermal-stressed European eel Ž Anguilla anguilla.. When fish were kept in 108C water, all activities increased to reach maximum levels at Day 8 with varying increasing rates of 3.0 Žproteolysis., 4.2 Žlysis of FlaÕobacterium columnare., 5.7 Žlysis of Vibrio anguillarum. and 7.8 Žlysis of Edwardsiella tarda. fold. High water temperature at 308C also induced marked increases in these activities, and the increasing rates were evaluated as 1.5 Žbacteriolysis. –2.0 Žproteolysis. fold higher than those at 108C. Results show a high sensitivity of eel skin cathepsins in bacteriolysis and proteolysis to thermal stress. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Thermal stress; Cathepsins L and B; Bacteriolysis; Skin; Anguilla anguilla
1. Introduction The aquatic environment is rich in pathogenic bacteria that often produce severe disease in fish ŽAustin and Austin, 1993.. Bacterial fish disease is a global problem in fish farming industry, because it is of major significance when fish are under stressful conditions in aquaculture ŽIwama et al., 1997.. Although bacterial fish disease occurs over a wide range of water temperature, bacteriosis is generally enhanced with rising temperature ŽThune et al., 1993.. The thermal effect on fish antibacterial defense therefore needs to be clarified in aquaculture ŽBly et al., 1997.. Anguilliformes, that are extensively farmed species in Asia and Europe, retain novel antibacterial proteases in the skin ŽAranishi and Nakane, 1997a,b.. These proteases, )
Tel.: q81-45-7887645; fax: q81-45-7885001; E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 7 5 - 6
210
F. Aranishir Aquaculture 182 (2000) 209–213
identified as cathepsins L and B, are secreted by skin epidermal cells ŽAranishi et al., 1998a. and catalyze lysis of bacteria ŽAranishi, 1999a,b.. Their activities significantly increase during bacteriosis via the skin ŽAranishi et al., 1998b., but the thermal effects on them remain unclear. The present study verifies bacteriolytic and proteolytic activities of skin cathepsins L and B in thermal-stressed European eel, Anguilla anguilla.
2. Materials and methods 2.1. Experimental procedure Adult European eel, reared to a mean weight of 180 g, were obtained from a commercial fish farm and kept in a 60 l well-aerated and filtered laboratory aquarium at 20.0 " 0.58C over 60 days to allow for acclimation and evaluation of fish health. The experiment on thermal stress employed two groups of five fish that were kept separately at 10.0 " 0.88C and 30.0 " 1.08C for up to 10 days ŽAranishi et al., 1998b.. During the experimental period, the dorsal skin was sampled from a fish at an area of 2 cm2 without overlap every other day and used in prepararing the skin extract ŽAranishi and Nakane, 1997b.. Protein concentration of the extract was measured by a DC Protein Microassay ŽBio-Rad.. 2.2. Bacteriolytic assay Aliquots of the skin extract were incubated with formalin-killed FlaÕobacterium columnare, Vibrio anguillarum or Edwardsiella tarda in 10 mM sodium acetate buffer, pH 5.0, at 378C for 10 min then 208C for 5 min ŽAranishi, 1999a.. The turbidity of the assay mixture was measured at 550 nm before and after reaction Ž1 unit s 0.01 turbidity decreasermin.. 2.3. Proteolytic assay Aliquots of the skin extract were incubated with 2 mM carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide ŽPeptide Institute. in 80 mM sodium acetate buffer, pH 5.0, at 378C for 10 min ŽAranishi and Nakane, 1997a.. The fluorescence of 7-amino-4-methylcoumarin ŽAMC. was measured at an excitation of 380 nm and emission of 460 nm Ž1 unit s 1 nmol AMC liberationrmin..
3. Results 3.1. Low water temperature Fig. 1 shows increases in the activities to lyse three species of pathogenic bacteria in the skin of European eel kept at 108C. The increase in the lysis of E. tarda proceeded
F. Aranishir Aquaculture 182 (2000) 209–213
211
Fig. 1. Skin catheptic bacteriolysis of F. columnare ŽA., V. anguillarum ŽB. or E. tarda ŽC. and proteolysis ŽD. in European eel at 108C Žmean"S.D., ns 5..
linearly up to Day 4, and the lysis of F. columnare or V. anguillarum increased at a high or low rate, respectively. These activities reached similar levels at Day 6, and their maximum increases commonly occurred from Day 6 to Day 8. The increasing rates from Day 2 to Day 8 were evaluated to be F. columnare- V. anguillarum- E. tarda between 4.2 and 7.8-fold. The decreases in the lysis of E. tarda and V. anguillarum after Day 8 were larger than the decrease in the lysis of F. columnare. As well as the bacteriolytic activities, the proteolytic activity increased from Day 2 to Day 8 and then decreased. Its increasing rate from Day 2 to Day 8 was evaluated to be 3.0-fold and less than the increasing rates of the bacteriolytic activities. 3.2. High water temperature The changing profiles of the bacteriolytic activities in the eel skin kept at 308C resembled those at 108C and consequently showed a high sensitivity of skin cathepsins to thermal stress. All of the bacteriolytic activities also reached maximum levels at Day 8, and the order of the increasing rates was the same as that at 108C ŽFig. 2.. Their maximum levels were, however, estimated as 1.5-fold higher than those at 108C. The
212
F. Aranishir Aquaculture 182 (2000) 209–213
Fig. 2. Skin catheptic bacteriolysis of F. columnare ŽA., V. anguillarum ŽB. or E. tarda ŽC. and proteolysis ŽD. in European eel at 308C Žmean"S.D., ns 5..
thermal stress of high water temperature therefore stimulated skin catheptic bacteriolysis more intensely than that of low water temperature. This appeared to be similar for the proteolytic activity, and the increasing rate from Day 2 to Day 8 was equivalent to 2.0-fold higher than at 108C.
4. Discussion This study first demonstrates the high sensitivity of eel skin cathepsins L and B in bacteriolysis and proteolysis to thermal stress. It suggests that their host defense ability is accelerated under stressful conditions, in which bacteriosis frequently occurs. An array of host defense substances in fish skin are either individually or cooperatively stimulated to respond to a variety of environmental stress, such as temperature, osmosis, pollution and density, in aquaculture facilities ŽRice et al., 1996; Bly et al., 1997; Iwama et al., 1997.. In particular, thermal stress is of great significance in bacterial infection, because bacterial disease occurs over a wide range of water temperature ŽThune et al., 1993..
F. Aranishir Aquaculture 182 (2000) 209–213
213
European eel is usually reared around 208C in Japanese aquaculture facilities. In parallel with the thermal-stressed experiments presented in this paper, no significant alteration was observed in various defense activities in the eel skin including both bacteriolytic and proteolytic activities due to cathepsins L and B, when fish were kept at 20.0 " 0.58C under the laboratory conditions Žnot shown.. However, Figs. 1 and 2 show marked increases in these catheptic activities within a few days at both 108C and 308C. These results indicate that an activation mechanism of eel skin cathepsins, probably depending on maturation from their precursory forms, is very susceptible to thermal stress. It is likely that the enhancement of potential catheptic activities in fish skin to respond to thermal stress could contribute to that of resisting ability of fish to bacterial infection. Three bacterial species employed as substrates for eel skin cathepsins are all potential pathogens with extensive fresh-water fish including Anguilliformes ŽAustin and Austin, 1993.. It is known that the bacterial fish pathogens of major importance are, with few exceptions, gram-negative, which have the membrane on the outside of the proteoglycan wall. Aranishi Ž1999a. reports that skin cathepsins L and B of Japanese eel, Anguilla japonica, catalyze lysis of V. anguillarum in an acidic region, and their catalytic action depends on the degradation of specific proteins of the bacterial outer membrane ŽAranishi, 1998.. In this study, the lysis of F. columnare and E. tarda in addition to V. anguillarum is likely to be caused by the same proteolytic action of skin cathepsins of European eel on the bacterial outer membrane, because of the synchronized changes of proteolytic with bacteriolytic activities.
References Aranishi, F., 1998. Cathepsins B and L responsible for defense bacteriolysis in Anguilliform skin. Proc. Int. Symp. Aquat. Anim. Health 3, 222. Aranishi, F., 1999a. Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel. Fish Physiol. Biochem. 20, 37–41. Aranishi, F., 1999b. Possible role for cathepsins B and L in bacteriolysis by Japanese eel skin. Fish Shellfish Immunol. 9, 61–64. Aranishi, F., Nakane, M., 1997a. Epidermal proteases of the Japanese eel. Fish Physiol. Biochem. 16, 471–478. Aranishi, F., Nakane, M., 1997b. Epidermal proteinases in European eel. Physiol. Zool. 70, 563–570. Aranishi, F., Mano, N., Hirose, H., 1998a. Fluorescence localization of epidermal cathepsins L and B in the Japanese eel. Fish Physiol. Biochem. 19, 205–209. Aranishi, F., Mano, N., Nakane, M., Hirose, H., 1998b. Epidermal response of the Japanese eel to environmental stress. Fish Physiol. Biochem. 19, 197–203. Austin, B., Austin, D.A., 1993. Diseases in farmed and wild fish. In: Laird, L.M. ŽEd.., Bacterial Fish Pathogens. Ellis Horwood, Chichester, pp. 263–296. Bly, J.E., Quiniou, S.M., Clem, L.W., 1997. Environmental effects on fish immune mechanisms. Dev. Biol. Stand. 90, 33–43. Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B., 1997. Fish Stress and Health in Aquaculture. Cambridge Univ. Press, Cambridge. Rice, C.D., Kergosien, D.H., Adams, S.M., 1996. Innate immune function as a bioindicator of pollution stress in fish. Ecotoxicol. Environ. Saf. 33, 186–192. Thune, R.L., Stanley, L.A., Cooper, K., 1993. Pathogenesis of gram-negative bacterial infections in warm water fish. Annu. Rev. Fish Dis. 3, 37–68.