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Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress
Veterinary Immunology and Immunopathology 89 (2002) 29–36
Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress Jesu´s Ortun˜o, M. Angeles Esteban, J. Meseguer* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain Received 27 December 2001; received in revised form 23 April 2002; accepted 13 May 2002
Abstract Phenoxyethanol is routinely used in seabream aquaculture to minimise fish stress response despite the secondary negative effects which have been observed. In this study, two different doses (60 and 200 ml/l) of phenoxyethanol, sedative and narcotic, were tested for their ability to reduce the stress caused in gilthead seabream (Sparus aurata L.) by crowding. Blood glucose and serum cortisol concentrations were measured as stress indicators. In order to study the effects of the treatment on the innate immune system of crowded specimens, two parameters of the innate immune response, serum complement activity and phagocytosis, were assessed. The results show that anaesthesia itself produced a stress response in the fish and affected the immune system, although the effects were greater with the narcotic dose. When the effects of anaesthesia on crowded fish were analysed, the results pointed to a slight reduction in stress as a result of the sedative dose of phenoxyethanol (lower increase in cortisol and lower reduction in phagocytosis). However, additive negative effects were seen in crowded fish when the narcotic dose of phenoxyethanol was used. Since the use of phenoxyethanol is a common practice in aquaculture, the significance of the results should be considered. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Phenoxyethanol; Sedative and narcotic doses; Glucose; Cortisol; Stress response; Phagocytosis; Gilthead seabream (Sparus aurata L.)
1. Introduction Although phenoxyethanol is considered as a general anaesthetic agent (Burka et al., 1997), and a suitable anaesthetic for fish (Idler et al., 1961), its mode of action is poorly understood. It acts rapidly and, when exposure is limited, recovery is also good and rapid. Moreover, its easy preparation and its low cost makes *
Corresponding author. Tel.: þ34-968364965; fax: þ34-968363963. E-mail address: [email protected] (J. Meseguer).
this chemical very suitable for aquacultural practices (Puce´at et al., 1989), fish farmers preferring it to other common chemicals such as tricaine or quinaldine sulphate. Phenoxyethanol is used in seabream aquaculture to minimise the fish stress response despite the secondary negative effects which are sometimes observed, and the fact that its usefulness has been questioned (Wedemeyer, 1997; Pickering, 1998). In some cases, such as severe anaesthesia, phenoxyethanol itself may induce a stress response in fish (Iwama et al., 1989; Thomas and Robertson, 1991; Ortun˜o et al., 2002), and stress is
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 1 8 3 - 6
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known to be a risk factor that may lead to immunodepression, thus facilitating subsequent infections (Balm, 1997; Pickering, 1998). While immunodepressive effects caused by anaesthesia have frequently been described in mammals (Rem et al., 1980; Whelan and Morris, 1982; Bardosi et al., 1990, 1992; Fescharek et al., 1994), there is little information on this topic in fish (Ortun˜ o et al., 2002). The aim of the present work was to study the possible secondary effects of phenoxyethanol on the innate immune system when used to reduce the stress response caused by crowding in gilthead seabream (Sparus aurata L.). With this purpose, blood glucose and serum cortisol concentrations were measured as stress indicators, while serum complement activity and phagocytosis of head-kidney leucocytes were assessed as parameters of the innate immune system.
2. Materials and methods 2.1. Fish Thirty-six specimens (150 16 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (S. aurata L.), obtained from Culmarex S.A. (Murcia, Spain), were kept in six running seawater (salinity 22%) aquaria (flow rate 1500 l/h), at 20 2 8C and with a natural photoperiod. All the fish were fed 1% body weight of commercial dry pellets (ProAqua Nutricio´ n S.A., Palencia, Spain) and allowed to acclimatise for 30 days before the experiments. 2.2. Experimental design Six experimental groups of six fish each were established. Group 1 was maintained at a standard fish density of 9 kg/m3, without anaesthesia, as negative control; group 2 was maintained at 100 kg/m3 (crowding) without anaesthesia, as positive control; groups 3 and 4 were maintained at 9 kg/m3 and were anaesthetised with 60 and 200 ml/l 2-phenoxyethanol (Sigma), respectively; groups 5 and 6 were maintained at 100 kg/m3 and anaesthetised with 60 and 200 ml/l 2phenoxyethanol, respectively. Fish were exposed to anaesthesia and/or crowding for 1 h. The dosages of phenoxyethanol tested in the present paper were chosen according to those used for goldfish (Josa et al.,
1992). The anaesthetic was administered by dissolving the appropriate quantity in a small portion of sea water and shaking, and then pouring the mixture into the aquaria. The increase in fish density was reached by reducing the volume of the water and maintaining the water-dissolved oxygen concentration. 2.3. Samples After experiments, 2 ml blood samples were collected from the caudal vein of each specimen using a 27-gauge needle and 1 ml syringe. Aliquots of 0.05 ml of those fresh blood samples were used for glucose analysis. The rest of each blood sample was allowed to clot at room temperature for 4 h. Following centrifugation, the serum was removed and frozen at 80 8C until the natural complement activity was determined. Head-kidney leucocytes were isolated according to Esteban et al. (1998). The head-kidney was dissected out by a ventral incision, cut into small fragments and transferred to 8 ml sRPMI-1640 medium: RPMI-1640 medium supplemented with 10 IU/ml heparin (Sigma), 100 IU/ml penicillin (Biochrom), 100 mg/ml streptomycin (Biochrom), 2% foetal calf serum (Gibco) and 0.35% sodium chloride (to adjust the osmolarity of the medium to seabream plasma osmolarity). Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 mm). Headkidney cell suspensions were layered over a 34– 51% Percoll density gradient (Pharmacia) and centrifuged at 400 g for 30 min at 4 8C. After centrifugation, the band of leucocytes above the 34–51% interface was collected with a Pasteur pipette, washed twice, counted and adjusted to 107 cells/ml in sRPMI. Cell viability was greater than 95%, as determined by the trypan blue exclusion test. 2.4. Stress indicators Samples of 0.05 ml fresh blood were used to measure glucose levels by means of a commercial kit based on the glucose dehydrogenase enzyme (Boehringer Mannheim). Cortisol levels were analysed in 100 ml serum samples by means of the TDx/TDxFLx Cortisol assay (Abbott Laboratories), which uses fluorescence polarisation immunoassay technology. The cortisol present in the samples competes with labelled cortisol to bind
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with anti-cortisol monoclonal antibody. Fluorescence changes were detected in a previously calibrated analyser (TDxFLx analyzer, Abbott Laboratories) (Bentley, 1976). 2.5. Natural complement activity The activity of the alternative complement pathway was assayed using sheep red blood cells (SRBC, Biomedics) as targets (Ortun˜ o et al., 1998). SRBC were washed in phenol red-free Hank’s buffer (HBSS) containing Mg2þ and EGTA and resuspended at 3% (v/v) in HBSS. Aliquots of 100 ml of test serum as complement source, diluted in HBSS, was added to 100 ml of SRBC in a flat-bottomed 96-well plate to give final serum concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.1565 and 0.078%. After incubation for 1 h at 22 8C, the samples were centrifuged at 400 g for 5 min at 4 8C to avoid unlysed erythrocytes. The relative haemoglobin content of the supernatants was assessed by measuring their optical density at 540 nm in a fluorimeter (BMG, Fluoro Star Galaxy). The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 100 ml of distilled water or HBSS to 100 ml samples of SRBC, respectively. Control samples using heat-inactivated serum were also added in each assay. The degree of haemolysis (Y) (percentage of haemolytic activity with respect to the maximum) was estimated and the lysis curve for each specimen was obtained by plotting Y=ð1 YÞ against the volume of serum added (ml) on a log10–log10 scaled graph. The volume of serum producing 50% haemolysis (ACH50) was determined and the number of ACH50 units/ml was obtained for each experimental group. 2.6. Phagocytic activity The phagocytic activity of gilthead seabream headkidney leucocytes was studied by flow cytometry according to Esteban et al. (1998). Vibrio anguillarum strain R82 (serotype 01) (Toranzo and Barja, 1990) was previously labelled with fluorescein isothiocyanate (FITC) (Sigma) as previously reported (Esteban et al., 1998) and used as test particle. Samples of 100 ml head-kidney leucocyte suspensions (previously adjusted to 107 cells/ml in sRPMI1640) were mixed with 10 ml FITC-labelled bacteria
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(previously adjusted to 109 bacteria/ml in PBS) and the samples were centrifuged (400 g, 5 min, 22 8C). Afterwards, the samples were resuspended and incubated at 22 8C for 30 min. At the end of the incubation time the samples were placed on ice to stop phagocytosis and 500 ml ice-cold PBS were added to each sample. The fluorescence of the extracellular bacteria was quenched by adding 10 ml ice-cold trypan blue (0.4% in PBS) per sample. Immediately, the samples were mixed gently and analysed in the flow cytometer. Standard samples of FITC-labelled V. anguillarum cells or seabream leucocytes were included in each phagocytosis assay. Samples incubated at 4 8C were used as negative controls. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria (green-fluorescent cells) within the total cell population (10,000 cells). 2.7. Statistical analysis All assays were performed in triplicate and the mean standard error (S.E.) calculated for each group ðn ¼ 6Þ. The data from the flow cytometric phagocytic assay were studied by using the statistical option of the Lysis Software Package (Becton Dickinson). Data were analysed statistically by one-way analysis of variance (ANOVA) to observe any difference due to treatment. Bonferroni’s test was used to determine differences between groups. Differences were considered statistically significant when P < 0:05.
3. Results 3.1. Blood glucose concentration The blood glucose concentration of control groups (resting and crowded fish not exposed to anaesthesia), were 40 4 and 112 7 mg/dl, respectively. When resting fish were anaesthetised with the two doses tested, they showed blood glucose values similar to those of the crowded control group. Crowded fish anaesthetised with 60 ml/l phenoxyethanol showed glucose values similar to those of the crowded control fish, while crowded fish anaesthetised with 200 ml/l showed glucose values double those observed in crowded control fish or crowded fish anaesthetised with 60 ml/l phenoxyethanol.
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Fig. 1. Blood glucose concentration of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
Fig. 2. Serum cortisol concentration of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
An ANOVA test pointed to statistically significant differences between the groups due to the treatment used ðP < 0:05Þ. Bonferroni’s test showed three different categories of significance: (a) a group showing the lowest glucose value (non-anaesthetised resting fish); (c) a group showing the highest glucose value (200 ml/l phenoxyethanol-anaesthetised crowded fish); (b) and the other four groups showing moderate glucose values (Fig. 1).
the lowest cortisol value (non-anaesthetised resting fish); (b) three groups with very high cortisol values (200 ml/l phenoxyethanol-anaesthetised resting fish, non-anaesthetised crowded fish and 200 ml/l phenoxyethanol-anaesthetised crowded fish). The other two groups (60 ml/l phenoxyethanol-anaesthetised resting or crowded fish) showed moderate cortisol values with a significance level similar to levels a and b (Fig. 2). 3.3. Natural complement activity
3.2. Serum cortisol concentration The serum cortisol concentration of resting and crowded control groups, was 1:25 0:2 and 36:7 5 ng/ml, respectively. When resting or crowded fish were anaesthetised, the cortisol values depended on the dose of phenoxyethanol used. Resting or crowded fish anaesthetised with 60 ml/l showed cortisol values between those of the two controls. Resting or crowded fish anaesthetised with 200 ml/l phenoxyethanol showed cortisol values similar to crowded control fish. ANOVA test showed statistically significant differences between the groups due to the treatment ðP < 0:05Þ, while Bonferroni’s test showed two different categories of significance: (a) a group with
Serum complement activity, as measured by the mean number of ACH50 units/ml serum, was significantly depressed by crowding and/or anaesthesia. Crowded fish anaesthetised with 200 ml/l 2-phenoxyethanol showed an especially low complement titer, which was lower than the rest of the values by a statistically significant extent. ANOVA pointed to statistically significant differences between the groups due to the treatment used ðP < 0:01Þ. Bonferroni’s test showed three different categories of significance: (a) a group showing the highest complement titer (non-anaesthetised resting fish); (b) two groups showing middle complement titers (non-anaethetised crowded fish and 60 ml/l
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Fig. 3. Serum natural complement activity of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
Fig. 4. Phagocytic ability of head-kidney leucocytes from resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
phenoxyethanol-anaesthetised resting fish); (c) a group showing the lowest complement titer (200 ml/l phenoxyethanol-anaesthetised crowded fish). The rest two groups (200 ml/l phenoxyethanol-anaesthetised resting fish and 60 ml/l phenoxyethanol-anaesthetised crowded fish) showed complement titers with a significance level similar to levels b and c (Fig. 3).
with the lowest complement titer (200 ml/l phenoxyethanol-anaesthetised resting fish). The phagocytic activity of a fourth group (200 ml/l phenoxyethanolanaesthetised crowded fish) had a significance level similar to levels b and c (Fig. 4).
4. Discussion 3.4. Phagocytic activiy The phagocytic ability (percentage of phagocytic cells) of gilthead seabream head-kidney leucocytes was significantly lower in the crowded control group than in the resting control group. When resting or crowded fish were anaesthetised this activity was depressed only with the narcotic dose of phenoxyethanol. ANOVA test showed statistically significant differences between the groups due to the treatment used ðP < 0:05Þ. Bonferroni’s test showed three different categories of significance: (a) three groups with a high phagocytic activity (non-anaesthetised resting fish and 60 ml/l phenoxyethanol-anaesthetised resting or crowded fish); (b) a group with a moderate phagocytic activity (non-anaesthetised crowded fish); (c) a group
Anaesthesia is commonly used in aquaculture to reduce the negative effects of several kinds of stressor. However, the effectiveness of anaesthetics to minimise the fish stress response and the possible appearance of secondary effects have been poorly studied. In the present work, we attempt to simulate fish farm practice, using commonly used fish densities and phenoxyethanol doses. Previous studies suggest that several anaesthetics are able to reduce the fish stress response when suitably administered (Thomas and Robertson, 1991; Wedemeyer, 1997). However, two aspects observed in fish and other vertebrates raise the possibility that, on occasions, the cure may be worse than the underlying illness. Firstly, it has been reported that anaesthetics may themselves induce a stress response in fish, which
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suffer a rise in blood glucose levels when exposure time is too long (Iwama et al., 1989; Thomas and Robertson, 1991). Secondly, anaesthesia may have immunodepressive effects in mammals (Rem et al., 1980; Whelan and Morris, 1982; Bardosi et al., 1990, 1992; Fescharek et al., 1994) and also in fish (Ortun˜ o et al., 2002). These two considerations mean that a deeper study than the purely visual observation of the animals frequently made in fish farms is necessary. To our knowledge, no studies focus on the optimal dosages and efficacy of phenoxyethanol in seabream. For this reason, we used dosages similar to those described as optimal for rainbow trout (O. mykiss) and goldfish (Cyprinus carpio). In rainbow trout of 73 g mean weight, a dose of 200 ml/l phenoxyethanol was reported to be optimal for inducing narcotic anaesthesia (Gilderhus and Marking, 1987). In adult goldfish, 100 or 200 ml/l phenoxyethanol were adequate for long anaesthetic periods, while doses above 500 ml/l were considered potentially dangerous (Josa et al., 1992). To find adequate sedative and narcotic doses for seabream, a preliminary experiment was made in our laboratory, the result of which led us to establish the mentioned 60 and 200 ml/l, respectively. The criteria used for establishing the different stages of anaesthesia were obtained from Stoskopf (1993). Fish densities were studied in a previous paper, in which it was demonstrated that a fish density of 100 kg/m3 increases blood glucose and serum cortisol levels and decreases complement and leucocyte phagocytic activities (Ortun˜ o et al., 2001). For this reason, we used this high stock fish density as positive control of stress and immunodepression, and the mentioned activities as indicators of possible changes that could be brought about in the seabream innate immune system by phenoxyethanol treatment. Blood glucose and serum cortisol concentrations are commonly used stress indicators in fish studies (reviewed by Pickering, 1998), and many studies have demonstrated that anaesthetics influence the magnitude of corticosteroid and/or hyperglycaemic responses of fish to stress (reviewed by Barton, 1997). In the present study, phenoxyethanol produced increases of glucose in resting fish equivalent to the increases observed in crowded fish. These data suggest that phenoxyethanol itself induces a stress response in seabream even at a sedative dose, although this seemed to have some
advantages compared with the narcotic dose. The sedative dose did not lead to any further increase in glucose when used on crowded fish, while the narcotic dose did. In this sense, it has been demonstrated that various stressors can induce a multiple stress which is characterised by the appearance of additive or, even, synergistic effects on the fish physiological responses (Barton et al., 1986). As regards the cortisol values obtained in the present work, phenoxyethanol induced a stress response in the fish, whose intensity depended on the dose used. In resting fish, the sedative dose produced a lower increase in cortisol than that provoked by the narcotic dose, and even lower than that induced by crowding stress. Although the data are not totally convincing, the difference between the narcotic and sedative doses is clear. In crowded fish, the sedative dose (but not the narcotic dose) seemed to minimise the increase provoked by crowding in fish cortisol levels. Such a reduction in cortisol values by anaesthetics has been described in rainbow trout (Iwama et al., 1989). The narcotic dose of anaesthetic and crowding stress did not produce additive effects on cortisol concentration, as was the case for the glucose values. The present data regarding glucose and cortisol concentrations point to the appearance of a stress response induced by phenoxyethanol in gilthead seabream specimens, as has been described in other fish species using different anaesthetics (Lewis et al., 1985; Belanger et al., 1986; Quinn et al., 1988; Taylor, 1988) and certain advantages of light sedation as opposed to deep narcosis. Complement and phagocytic activities are suitable immune indicators, and are widely used in fish stress studies (Scott and Klesius, 1981; Ghoneum et al., 1988; Thompson et al., 1993; Yin et al., 1995; Tort et al., 1996; Ortun˜ o et al., 2001, 2002). In the present paper, seabream serum complement activity was depressed by crowding, the administration of phenoxyethanol or both. It has been previously described that the seabream serum haemolytic activity can be affected by a wide variety of factors, including short-term stressors (Tort et al., 1996; Ortun˜ o et al., 2001), while the effects of anaesthesia have usually been ignored. Indeed, there is little information available on the relationship between anaesthetics and the fish immune system. Just one previous work from our laboratory described depression of the complement activity by phenoxyethanol at 200 ml/l (Ortun˜ o et al., 2002).
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Immunodepressive effects have also been observed in mammals as a consequence of the administration of anaesthetics. In humans, epidural analgesia deeply depresses the concentration of complement components and their total haemolytic activity (Hauptmann et al., 1985; Gajdosz, 1994), effects which have been attributed to the extent of sympathetic interruption caused by the analgesia. In the present experiment, this mechanism of sympathetic interruption might have caused the depletion of haemolytic activity. Again, narcosis and crowding jointly applied have an additive effect, producing the highest depression in complement titers, which correlates with the results of the glucose concentrations. Gilthead seabream phagocytosis was depressed by phenoxyethanol at the narcotic but not at the sedative dose. In mammals, anaesthetic agents are known to depress phagocytic functions such as leucocyte recruitment, attachment, chemotactic motility, engulfment and intracellular killing (Bardosi et al., 1990). A progressive decline in the expression of endogenous sugar receptors, such as mannose receptors, in granulocytes during anaesthesia has been detected (Bardosi et al., 1990, 1992). Considering that carbohydrate–lectin interactions play an important role in the activities of phagocytic cells, the above author suggested that this depletion of lectin-like receptors could be the connection between anaesthesia and depression of the phagocytic function. In fish, lectin-like receptors also play an important role in the phagocytic function, allowing the interaction between the antigen and the phagocyte surface (Secombes, 1996), and a similar event might have occurred during the narcosis of the fish described in the present work, but not during the sedation of the fish. In addition, the sedative dose of the anaesthetic seemed to protect the phagocytic function against the depression caused by crowding. This result correlates with the amelioration by phenoxyethanol at a sedative dose of the crowding-mediated cortisol increase, and could be a reflection of this. The parallelism observed between glucose and complement results and between cortisol and phagocytosis suggests a specific relationship between a given stress mediator and a given immune activity. In this sense, adrenaline seems to have more effect on the complement system than on the phagocytic function, the opposite being true for cortisol.
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To conclude, under the specified experimental conditions, the anaesthetic 2-phenoxyethanol used at a narcotic dose of 200 ml/l induced a stress response and immunodepression in gilthead seabream, effects that were more severe when fish were exposed to crowding stress. However, when 2-phenoxyethanol was used at a sedative dose of 60 ml/l, fish showed a milder stress response and immunodepression and even certain protection against crowding stress. These results should be taken into account during processes such as classification, transport or manipulation, when crowded fish are frequently anaesthetised.
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Puce´ at, M., Garin, D., Fre´ minet, A., 1989. Inhibitory effect of anaesthesia with 2-phenoxyethanol as compared to MS222 on glucose release in isolated hepatocytes from rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. A 94, 221– 224. Quinn, T.P., Olson, A.F., Konecki, J.T., 1988. Effects of anesthesia on the chemosensory behaviour of Pacific salmon. J. Fish Biol. 33, 637–641. Rem, J., Brandt, M.R., Kehlet, H., 1980. Prevention of postoperative lymphopenia and granulocytosis by epidural analgesia. Lancet 1 (8163), 283–284. Scott, A., Klesius, P.H., 1981. Chemiluminescence: a novel analysis of phagocytosis in fish. In: Anderson, D.P., Hennessen, W. (Eds.), Developments in Biological Standardization, Vol. 49. S. Karger, Basel, pp. 243–256. Secombes, C.J., 1996. The innate immune system: cellular defences. In: Iwama, G., Nakanishi, T. (Eds.), The Fish Immune System. Academic Press, San Diego, CA, pp. 63– 103. Stoskopf, M.K., 1993. Anaesthesia. In: Brown, L. (Ed.), Aquaculture for Veterinarians: Fish Husbandry and Medicine. Pergamon Press, Oxford, pp. 161–167. Taylor, P.B., 1988. Effects of anaesthetic MS 222 on the orientation of juvenile salmon, Oncorhynchus tschawytscha Walbaum. J. Fish Biol. 32, 161–168. Thomas, P., Robertson, L., 1991. Plasma cortisol and glucose stress responses of red drum (Sciaenops ocellatus) to handling and shallow water stressors and anesthesia with MS-222, quinaldine sulphate and metomidate. Aquaculture 96, 69–86. Thompson, I., White, A., Fletcher, T.C., Houlihan, D.F., Secombes, C.J., 1993. The effect of stress on the immune system of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin C. Aquaculture 114, 1–18. Toranzo, A.E., Barja, J.L., 1990. A review of the taxonomy and seroepizootiology of Vibrio anguillarum, with special reference to aquaculture in the northwest of Spain. Dis. Aquacult. Org. 9, 73–82. Tort, L., Go´ mez, E., Montero, D., Sunyer, J.O., 1996. Serum haemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies. Aquacult. Int. 4, 31–41. Wedemeyer, G.A., 1997. Effects of rearing conditions on the health and physiological quality of fish in intensive culture. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.), Fish Stress and Health in Aquaculture. Cambridge University Press, Cambridge, pp. 35–72. Whelan, P., Morris, P.J., 1982. Immunological responsiveness after transurethral resection of the prostate: general versus spinal anaesthetic. Clin. Exp. Immunol. 48, 611–618. Yin, Z., Lam, T.J., Sin, Y.M., 1995. The effects of crowding stress on the non-specific immune response in fancy carp (Cyprinus carpio L.). Fish Shellfish Immunol. 5, 519–529.
Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress Jesu´s Ortun˜o, M. Angeles Esteban, J. Meseguer* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain Received 27 December 2001; received in revised form 23 April 2002; accepted 13 May 2002
Abstract Phenoxyethanol is routinely used in seabream aquaculture to minimise fish stress response despite the secondary negative effects which have been observed. In this study, two different doses (60 and 200 ml/l) of phenoxyethanol, sedative and narcotic, were tested for their ability to reduce the stress caused in gilthead seabream (Sparus aurata L.) by crowding. Blood glucose and serum cortisol concentrations were measured as stress indicators. In order to study the effects of the treatment on the innate immune system of crowded specimens, two parameters of the innate immune response, serum complement activity and phagocytosis, were assessed. The results show that anaesthesia itself produced a stress response in the fish and affected the immune system, although the effects were greater with the narcotic dose. When the effects of anaesthesia on crowded fish were analysed, the results pointed to a slight reduction in stress as a result of the sedative dose of phenoxyethanol (lower increase in cortisol and lower reduction in phagocytosis). However, additive negative effects were seen in crowded fish when the narcotic dose of phenoxyethanol was used. Since the use of phenoxyethanol is a common practice in aquaculture, the significance of the results should be considered. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Phenoxyethanol; Sedative and narcotic doses; Glucose; Cortisol; Stress response; Phagocytosis; Gilthead seabream (Sparus aurata L.)
1. Introduction Although phenoxyethanol is considered as a general anaesthetic agent (Burka et al., 1997), and a suitable anaesthetic for fish (Idler et al., 1961), its mode of action is poorly understood. It acts rapidly and, when exposure is limited, recovery is also good and rapid. Moreover, its easy preparation and its low cost makes *
Corresponding author. Tel.: þ34-968364965; fax: þ34-968363963. E-mail address: [email protected] (J. Meseguer).
this chemical very suitable for aquacultural practices (Puce´at et al., 1989), fish farmers preferring it to other common chemicals such as tricaine or quinaldine sulphate. Phenoxyethanol is used in seabream aquaculture to minimise the fish stress response despite the secondary negative effects which are sometimes observed, and the fact that its usefulness has been questioned (Wedemeyer, 1997; Pickering, 1998). In some cases, such as severe anaesthesia, phenoxyethanol itself may induce a stress response in fish (Iwama et al., 1989; Thomas and Robertson, 1991; Ortun˜o et al., 2002), and stress is
0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 1 8 3 - 6
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known to be a risk factor that may lead to immunodepression, thus facilitating subsequent infections (Balm, 1997; Pickering, 1998). While immunodepressive effects caused by anaesthesia have frequently been described in mammals (Rem et al., 1980; Whelan and Morris, 1982; Bardosi et al., 1990, 1992; Fescharek et al., 1994), there is little information on this topic in fish (Ortun˜ o et al., 2002). The aim of the present work was to study the possible secondary effects of phenoxyethanol on the innate immune system when used to reduce the stress response caused by crowding in gilthead seabream (Sparus aurata L.). With this purpose, blood glucose and serum cortisol concentrations were measured as stress indicators, while serum complement activity and phagocytosis of head-kidney leucocytes were assessed as parameters of the innate immune system.
2. Materials and methods 2.1. Fish Thirty-six specimens (150 16 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (S. aurata L.), obtained from Culmarex S.A. (Murcia, Spain), were kept in six running seawater (salinity 22%) aquaria (flow rate 1500 l/h), at 20 2 8C and with a natural photoperiod. All the fish were fed 1% body weight of commercial dry pellets (ProAqua Nutricio´ n S.A., Palencia, Spain) and allowed to acclimatise for 30 days before the experiments. 2.2. Experimental design Six experimental groups of six fish each were established. Group 1 was maintained at a standard fish density of 9 kg/m3, without anaesthesia, as negative control; group 2 was maintained at 100 kg/m3 (crowding) without anaesthesia, as positive control; groups 3 and 4 were maintained at 9 kg/m3 and were anaesthetised with 60 and 200 ml/l 2-phenoxyethanol (Sigma), respectively; groups 5 and 6 were maintained at 100 kg/m3 and anaesthetised with 60 and 200 ml/l 2phenoxyethanol, respectively. Fish were exposed to anaesthesia and/or crowding for 1 h. The dosages of phenoxyethanol tested in the present paper were chosen according to those used for goldfish (Josa et al.,
1992). The anaesthetic was administered by dissolving the appropriate quantity in a small portion of sea water and shaking, and then pouring the mixture into the aquaria. The increase in fish density was reached by reducing the volume of the water and maintaining the water-dissolved oxygen concentration. 2.3. Samples After experiments, 2 ml blood samples were collected from the caudal vein of each specimen using a 27-gauge needle and 1 ml syringe. Aliquots of 0.05 ml of those fresh blood samples were used for glucose analysis. The rest of each blood sample was allowed to clot at room temperature for 4 h. Following centrifugation, the serum was removed and frozen at 80 8C until the natural complement activity was determined. Head-kidney leucocytes were isolated according to Esteban et al. (1998). The head-kidney was dissected out by a ventral incision, cut into small fragments and transferred to 8 ml sRPMI-1640 medium: RPMI-1640 medium supplemented with 10 IU/ml heparin (Sigma), 100 IU/ml penicillin (Biochrom), 100 mg/ml streptomycin (Biochrom), 2% foetal calf serum (Gibco) and 0.35% sodium chloride (to adjust the osmolarity of the medium to seabream plasma osmolarity). Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 mm). Headkidney cell suspensions were layered over a 34– 51% Percoll density gradient (Pharmacia) and centrifuged at 400 g for 30 min at 4 8C. After centrifugation, the band of leucocytes above the 34–51% interface was collected with a Pasteur pipette, washed twice, counted and adjusted to 107 cells/ml in sRPMI. Cell viability was greater than 95%, as determined by the trypan blue exclusion test. 2.4. Stress indicators Samples of 0.05 ml fresh blood were used to measure glucose levels by means of a commercial kit based on the glucose dehydrogenase enzyme (Boehringer Mannheim). Cortisol levels were analysed in 100 ml serum samples by means of the TDx/TDxFLx Cortisol assay (Abbott Laboratories), which uses fluorescence polarisation immunoassay technology. The cortisol present in the samples competes with labelled cortisol to bind
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with anti-cortisol monoclonal antibody. Fluorescence changes were detected in a previously calibrated analyser (TDxFLx analyzer, Abbott Laboratories) (Bentley, 1976). 2.5. Natural complement activity The activity of the alternative complement pathway was assayed using sheep red blood cells (SRBC, Biomedics) as targets (Ortun˜ o et al., 1998). SRBC were washed in phenol red-free Hank’s buffer (HBSS) containing Mg2þ and EGTA and resuspended at 3% (v/v) in HBSS. Aliquots of 100 ml of test serum as complement source, diluted in HBSS, was added to 100 ml of SRBC in a flat-bottomed 96-well plate to give final serum concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.1565 and 0.078%. After incubation for 1 h at 22 8C, the samples were centrifuged at 400 g for 5 min at 4 8C to avoid unlysed erythrocytes. The relative haemoglobin content of the supernatants was assessed by measuring their optical density at 540 nm in a fluorimeter (BMG, Fluoro Star Galaxy). The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 100 ml of distilled water or HBSS to 100 ml samples of SRBC, respectively. Control samples using heat-inactivated serum were also added in each assay. The degree of haemolysis (Y) (percentage of haemolytic activity with respect to the maximum) was estimated and the lysis curve for each specimen was obtained by plotting Y=ð1 YÞ against the volume of serum added (ml) on a log10–log10 scaled graph. The volume of serum producing 50% haemolysis (ACH50) was determined and the number of ACH50 units/ml was obtained for each experimental group. 2.6. Phagocytic activity The phagocytic activity of gilthead seabream headkidney leucocytes was studied by flow cytometry according to Esteban et al. (1998). Vibrio anguillarum strain R82 (serotype 01) (Toranzo and Barja, 1990) was previously labelled with fluorescein isothiocyanate (FITC) (Sigma) as previously reported (Esteban et al., 1998) and used as test particle. Samples of 100 ml head-kidney leucocyte suspensions (previously adjusted to 107 cells/ml in sRPMI1640) were mixed with 10 ml FITC-labelled bacteria
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(previously adjusted to 109 bacteria/ml in PBS) and the samples were centrifuged (400 g, 5 min, 22 8C). Afterwards, the samples were resuspended and incubated at 22 8C for 30 min. At the end of the incubation time the samples were placed on ice to stop phagocytosis and 500 ml ice-cold PBS were added to each sample. The fluorescence of the extracellular bacteria was quenched by adding 10 ml ice-cold trypan blue (0.4% in PBS) per sample. Immediately, the samples were mixed gently and analysed in the flow cytometer. Standard samples of FITC-labelled V. anguillarum cells or seabream leucocytes were included in each phagocytosis assay. Samples incubated at 4 8C were used as negative controls. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria (green-fluorescent cells) within the total cell population (10,000 cells). 2.7. Statistical analysis All assays were performed in triplicate and the mean standard error (S.E.) calculated for each group ðn ¼ 6Þ. The data from the flow cytometric phagocytic assay were studied by using the statistical option of the Lysis Software Package (Becton Dickinson). Data were analysed statistically by one-way analysis of variance (ANOVA) to observe any difference due to treatment. Bonferroni’s test was used to determine differences between groups. Differences were considered statistically significant when P < 0:05.
3. Results 3.1. Blood glucose concentration The blood glucose concentration of control groups (resting and crowded fish not exposed to anaesthesia), were 40 4 and 112 7 mg/dl, respectively. When resting fish were anaesthetised with the two doses tested, they showed blood glucose values similar to those of the crowded control group. Crowded fish anaesthetised with 60 ml/l phenoxyethanol showed glucose values similar to those of the crowded control fish, while crowded fish anaesthetised with 200 ml/l showed glucose values double those observed in crowded control fish or crowded fish anaesthetised with 60 ml/l phenoxyethanol.
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Fig. 1. Blood glucose concentration of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
Fig. 2. Serum cortisol concentration of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
An ANOVA test pointed to statistically significant differences between the groups due to the treatment used ðP < 0:05Þ. Bonferroni’s test showed three different categories of significance: (a) a group showing the lowest glucose value (non-anaesthetised resting fish); (c) a group showing the highest glucose value (200 ml/l phenoxyethanol-anaesthetised crowded fish); (b) and the other four groups showing moderate glucose values (Fig. 1).
the lowest cortisol value (non-anaesthetised resting fish); (b) three groups with very high cortisol values (200 ml/l phenoxyethanol-anaesthetised resting fish, non-anaesthetised crowded fish and 200 ml/l phenoxyethanol-anaesthetised crowded fish). The other two groups (60 ml/l phenoxyethanol-anaesthetised resting or crowded fish) showed moderate cortisol values with a significance level similar to levels a and b (Fig. 2). 3.3. Natural complement activity
3.2. Serum cortisol concentration The serum cortisol concentration of resting and crowded control groups, was 1:25 0:2 and 36:7 5 ng/ml, respectively. When resting or crowded fish were anaesthetised, the cortisol values depended on the dose of phenoxyethanol used. Resting or crowded fish anaesthetised with 60 ml/l showed cortisol values between those of the two controls. Resting or crowded fish anaesthetised with 200 ml/l phenoxyethanol showed cortisol values similar to crowded control fish. ANOVA test showed statistically significant differences between the groups due to the treatment ðP < 0:05Þ, while Bonferroni’s test showed two different categories of significance: (a) a group with
Serum complement activity, as measured by the mean number of ACH50 units/ml serum, was significantly depressed by crowding and/or anaesthesia. Crowded fish anaesthetised with 200 ml/l 2-phenoxyethanol showed an especially low complement titer, which was lower than the rest of the values by a statistically significant extent. ANOVA pointed to statistically significant differences between the groups due to the treatment used ðP < 0:01Þ. Bonferroni’s test showed three different categories of significance: (a) a group showing the highest complement titer (non-anaesthetised resting fish); (b) two groups showing middle complement titers (non-anaethetised crowded fish and 60 ml/l
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Fig. 3. Serum natural complement activity of resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
Fig. 4. Phagocytic ability of head-kidney leucocytes from resting or crowded gilthead seabream specimens anaesthetised with 60 ml/l (&) or 200 ml/l (&) 2-phenoxyethanol. Data represent the mean S:E: The mean value belonging to resting or crowded control fish is represented by a discontinuous or continuous line, respectively. Different letters denote statistically significant differences between the groups (explained in the text).
phenoxyethanol-anaesthetised resting fish); (c) a group showing the lowest complement titer (200 ml/l phenoxyethanol-anaesthetised crowded fish). The rest two groups (200 ml/l phenoxyethanol-anaesthetised resting fish and 60 ml/l phenoxyethanol-anaesthetised crowded fish) showed complement titers with a significance level similar to levels b and c (Fig. 3).
with the lowest complement titer (200 ml/l phenoxyethanol-anaesthetised resting fish). The phagocytic activity of a fourth group (200 ml/l phenoxyethanolanaesthetised crowded fish) had a significance level similar to levels b and c (Fig. 4).
4. Discussion 3.4. Phagocytic activiy The phagocytic ability (percentage of phagocytic cells) of gilthead seabream head-kidney leucocytes was significantly lower in the crowded control group than in the resting control group. When resting or crowded fish were anaesthetised this activity was depressed only with the narcotic dose of phenoxyethanol. ANOVA test showed statistically significant differences between the groups due to the treatment used ðP < 0:05Þ. Bonferroni’s test showed three different categories of significance: (a) three groups with a high phagocytic activity (non-anaesthetised resting fish and 60 ml/l phenoxyethanol-anaesthetised resting or crowded fish); (b) a group with a moderate phagocytic activity (non-anaesthetised crowded fish); (c) a group
Anaesthesia is commonly used in aquaculture to reduce the negative effects of several kinds of stressor. However, the effectiveness of anaesthetics to minimise the fish stress response and the possible appearance of secondary effects have been poorly studied. In the present work, we attempt to simulate fish farm practice, using commonly used fish densities and phenoxyethanol doses. Previous studies suggest that several anaesthetics are able to reduce the fish stress response when suitably administered (Thomas and Robertson, 1991; Wedemeyer, 1997). However, two aspects observed in fish and other vertebrates raise the possibility that, on occasions, the cure may be worse than the underlying illness. Firstly, it has been reported that anaesthetics may themselves induce a stress response in fish, which
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suffer a rise in blood glucose levels when exposure time is too long (Iwama et al., 1989; Thomas and Robertson, 1991). Secondly, anaesthesia may have immunodepressive effects in mammals (Rem et al., 1980; Whelan and Morris, 1982; Bardosi et al., 1990, 1992; Fescharek et al., 1994) and also in fish (Ortun˜ o et al., 2002). These two considerations mean that a deeper study than the purely visual observation of the animals frequently made in fish farms is necessary. To our knowledge, no studies focus on the optimal dosages and efficacy of phenoxyethanol in seabream. For this reason, we used dosages similar to those described as optimal for rainbow trout (O. mykiss) and goldfish (Cyprinus carpio). In rainbow trout of 73 g mean weight, a dose of 200 ml/l phenoxyethanol was reported to be optimal for inducing narcotic anaesthesia (Gilderhus and Marking, 1987). In adult goldfish, 100 or 200 ml/l phenoxyethanol were adequate for long anaesthetic periods, while doses above 500 ml/l were considered potentially dangerous (Josa et al., 1992). To find adequate sedative and narcotic doses for seabream, a preliminary experiment was made in our laboratory, the result of which led us to establish the mentioned 60 and 200 ml/l, respectively. The criteria used for establishing the different stages of anaesthesia were obtained from Stoskopf (1993). Fish densities were studied in a previous paper, in which it was demonstrated that a fish density of 100 kg/m3 increases blood glucose and serum cortisol levels and decreases complement and leucocyte phagocytic activities (Ortun˜ o et al., 2001). For this reason, we used this high stock fish density as positive control of stress and immunodepression, and the mentioned activities as indicators of possible changes that could be brought about in the seabream innate immune system by phenoxyethanol treatment. Blood glucose and serum cortisol concentrations are commonly used stress indicators in fish studies (reviewed by Pickering, 1998), and many studies have demonstrated that anaesthetics influence the magnitude of corticosteroid and/or hyperglycaemic responses of fish to stress (reviewed by Barton, 1997). In the present study, phenoxyethanol produced increases of glucose in resting fish equivalent to the increases observed in crowded fish. These data suggest that phenoxyethanol itself induces a stress response in seabream even at a sedative dose, although this seemed to have some
advantages compared with the narcotic dose. The sedative dose did not lead to any further increase in glucose when used on crowded fish, while the narcotic dose did. In this sense, it has been demonstrated that various stressors can induce a multiple stress which is characterised by the appearance of additive or, even, synergistic effects on the fish physiological responses (Barton et al., 1986). As regards the cortisol values obtained in the present work, phenoxyethanol induced a stress response in the fish, whose intensity depended on the dose used. In resting fish, the sedative dose produced a lower increase in cortisol than that provoked by the narcotic dose, and even lower than that induced by crowding stress. Although the data are not totally convincing, the difference between the narcotic and sedative doses is clear. In crowded fish, the sedative dose (but not the narcotic dose) seemed to minimise the increase provoked by crowding in fish cortisol levels. Such a reduction in cortisol values by anaesthetics has been described in rainbow trout (Iwama et al., 1989). The narcotic dose of anaesthetic and crowding stress did not produce additive effects on cortisol concentration, as was the case for the glucose values. The present data regarding glucose and cortisol concentrations point to the appearance of a stress response induced by phenoxyethanol in gilthead seabream specimens, as has been described in other fish species using different anaesthetics (Lewis et al., 1985; Belanger et al., 1986; Quinn et al., 1988; Taylor, 1988) and certain advantages of light sedation as opposed to deep narcosis. Complement and phagocytic activities are suitable immune indicators, and are widely used in fish stress studies (Scott and Klesius, 1981; Ghoneum et al., 1988; Thompson et al., 1993; Yin et al., 1995; Tort et al., 1996; Ortun˜ o et al., 2001, 2002). In the present paper, seabream serum complement activity was depressed by crowding, the administration of phenoxyethanol or both. It has been previously described that the seabream serum haemolytic activity can be affected by a wide variety of factors, including short-term stressors (Tort et al., 1996; Ortun˜ o et al., 2001), while the effects of anaesthesia have usually been ignored. Indeed, there is little information available on the relationship between anaesthetics and the fish immune system. Just one previous work from our laboratory described depression of the complement activity by phenoxyethanol at 200 ml/l (Ortun˜ o et al., 2002).
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Immunodepressive effects have also been observed in mammals as a consequence of the administration of anaesthetics. In humans, epidural analgesia deeply depresses the concentration of complement components and their total haemolytic activity (Hauptmann et al., 1985; Gajdosz, 1994), effects which have been attributed to the extent of sympathetic interruption caused by the analgesia. In the present experiment, this mechanism of sympathetic interruption might have caused the depletion of haemolytic activity. Again, narcosis and crowding jointly applied have an additive effect, producing the highest depression in complement titers, which correlates with the results of the glucose concentrations. Gilthead seabream phagocytosis was depressed by phenoxyethanol at the narcotic but not at the sedative dose. In mammals, anaesthetic agents are known to depress phagocytic functions such as leucocyte recruitment, attachment, chemotactic motility, engulfment and intracellular killing (Bardosi et al., 1990). A progressive decline in the expression of endogenous sugar receptors, such as mannose receptors, in granulocytes during anaesthesia has been detected (Bardosi et al., 1990, 1992). Considering that carbohydrate–lectin interactions play an important role in the activities of phagocytic cells, the above author suggested that this depletion of lectin-like receptors could be the connection between anaesthesia and depression of the phagocytic function. In fish, lectin-like receptors also play an important role in the phagocytic function, allowing the interaction between the antigen and the phagocyte surface (Secombes, 1996), and a similar event might have occurred during the narcosis of the fish described in the present work, but not during the sedation of the fish. In addition, the sedative dose of the anaesthetic seemed to protect the phagocytic function against the depression caused by crowding. This result correlates with the amelioration by phenoxyethanol at a sedative dose of the crowding-mediated cortisol increase, and could be a reflection of this. The parallelism observed between glucose and complement results and between cortisol and phagocytosis suggests a specific relationship between a given stress mediator and a given immune activity. In this sense, adrenaline seems to have more effect on the complement system than on the phagocytic function, the opposite being true for cortisol.
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To conclude, under the specified experimental conditions, the anaesthetic 2-phenoxyethanol used at a narcotic dose of 200 ml/l induced a stress response and immunodepression in gilthead seabream, effects that were more severe when fish were exposed to crowding stress. However, when 2-phenoxyethanol was used at a sedative dose of 60 ml/l, fish showed a milder stress response and immunodepression and even certain protection against crowding stress. These results should be taken into account during processes such as classification, transport or manipulation, when crowded fish are frequently anaesthetised.
Acknowledgements This work was supported by a project from ‘CICYT’, Spanish Ministry of Science and Technology (reference number PB98-0387). Mr. Ortun˜ o has a research grant from ‘Caja Murcia’ (Social and Cultural Section). References Balm, P.H.M., 1997. Immune–endocrine interactions. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.), Fish Stress and Health in Aquaculture. Cambridge University Press, Cambridge, pp. 195–222. Bardosi, L., Bardosi, A., Hendrys, M., Gabius, H.J., 1990. Reduced expression of mannose-specific receptors on murine peripheral blood polymorphonuclear leukocytes following prolonged anaesthesia with different inhalation agents. Acta Anaesthesiol. Scand. 34, 286–290. Bardosi, L., Bardosi, A., Gabius, H.J., 1992. Changes of expression of endogenous sugar receptors by polymorphonuclear leukocytes after prolonged anaesthesia surgery. Can. J. Anaesth. 39, 143–150. Barton, B.A., 1997. Stress in finfish: past, present and future—a historical perspective. In: Iwama, G.K., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.), Fish Stress and Health in Aquaculture. Cambridge University Press, Cambridge, pp. 1– 33. Barton, B.A., Schreck, C.B., Sigismondi, L.A., 1986. Multiple acute disturbance evokes cumulative physiological stress responses in juvenile chinook salmon. Trans. Am. Fish. Soc. 115, 245–251. Belanger, S.E., Schurr, K., Allen, D.J., Gohara, A.F., 1986. Effects of chrysotile asbestos on coho salmon and green sunfish: evidence of behavioral and pathological stress. Environ. Res. 39, 74–85. Bentley, P.J., 1976. Comparative Vertebrate Endocrinology. Cambridge University Press, New York.
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