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High sensitivity of skin cathepsins L and B of
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European eel Anguilla 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
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in thermal-stressed European eel 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 lysis of Fla. Ž Õobacterium columnare , 5.7 lysis of Vibrio anguillarum and 7.8. Ž . Žlysis of Edwardsiella tarda fold. High water temperature at 30. 8C also induced marked increases
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in these activities, and the increasing rates were evaluated as 1.5 bacteriolysis –2.0 proteolysis fold higher than those at 108C. Results show a high sensitivity of eel skin cathepsins in bacteriolysis and proteolysis to thermal stress.q2000 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
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disease in fish Austin 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
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conditions in aquaculture Iwama et al., 1997 . Although bacterial fish disease occurs over a wide range of water temperature, bacteriosis is generally enhanced with rising
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temperature Thune et al., 1993 . The thermal effect on fish antibacterial defense
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therefore needs to be clarified in aquaculture Bly et al., 1997 .
Anguilliformes, that are extensively farmed species in Asia and Europe, retain novel
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antibacterial proteases in the skin Aranishi and Nakane, 1997a,b . These proteases,
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on them remain unclear. The present study verifies bacteriolytic and proteolytic activi-ties 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
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at 10.0"0.88C and 30.0"1.08C for up to 10 days Aranishi et al., 1998b . During the
experimental period, the dorsal skin was sampled from a fish at an area of 2 cm2
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without overlap every other day and used in prepararing the skin extract Aranishi and
.
Nakane, 1997b . Protein concentration of the extract was measured by a DC Protein
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Microassay Bio-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,
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pH 5.0, at 378C for 10 min then 208C for 5 min Aranishi, 1999a . The turbidity of the
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assay mixture was measured at 550 nm before and after reaction 1 units0.01 turbidity
.
decreasermin .
2.3. Proteolytic assay
Aliquots of the skin extract were incubated with 2 mM carbobenzoxy-L
-phenyl-Ž .
alanyl-L-arginyl-4-methylcoumaryl-7-amide Peptide Institute in 80 mM sodium acetate
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buffer, pH 5.0, at 378C for 10 min Aranishi and Nakane, 1997a . The fluorescence of
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7-amino-4-methylcoumarin AMC was measured at an excitation of 380 nm and
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emission of 460 nm 1 units1 nmol AMC liberationrmin .
3. Results
3.1. Low water temperature
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Fig. 1. Skin catheptic bacteriolysis of F. columnare A , V. anguillarum B or E. tarda C and proteolysis
Ž .D in European eel at 108C meanŽ "S.D., ns5 ..
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
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8, and the order of the increasing rates was the same as that at 108C Fig. 2 . Their
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Fig. 2. Skin catheptic bacteriolysis of F. columnare A , V. anguillarum B or E. tarda C and proteolysis
Ž .D in European eel at 308C meanŽ "S.D., ns5 ..
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,
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pollution and density, in aquaculture facilities Rice et al., 1996; Bly et al., 1997; Iwama
.
et al., 1997 . In particular, thermal stress is of great significance in bacterial infection,
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because bacterial disease occurs over a wide range of water temperature Thune et al.,
.
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
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20.0"0.58C under the laboratory conditions not 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
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pathogens with extensive fresh-water fish including Anguilliformes Austin 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
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wall. Aranishi 1999a 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.
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