Fish & Shellfish Immunology (2001) 11, 187–197 doi:10.1006/fsim.2000.0304 Available online at http://www.idealibrary.com on
Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L.) innate immune response J. ORTUNx O, M. A. ESTEBAN
AND
J. MESEGUER*
Department of Cell Biology, Faculty of Biology, University of Murcia, 30071 Murcia, Spain (Received 7 July 1999, accepted after revision 28 August 2000) Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m 3 for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate e#ect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained una#ected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish. 2001 Academic Press
Key words:
short-term crowding stress, innate immune response, complement, phagocytosis, respiratory burst, cell tra$cking, gilthead seabream.
I. Introduction The physiological e#ects of environmental stressors in fish depend on the duration and on the intensity of such stressors (Mosconi et al., 1998; Pickering, 1998). An acute stress response can be induced in fish as a result of short-term mild stressful conditions (Demers & Bayne, 1977; Rotllant & Tort, 1997; Ruis & Bayne, 1997), while a chronic stress response is induced in fish as a result of long-term severe stressful conditions (Pickering, 1998). The impact of these two categories of stress (acute and chronic) on the fish immune system has been the object of much study in recent years. The enhancement of fish immune response, rather than its immunosuppression, has been observed to follow acute stress (increased concentrations of specific plasma proteins, such as lysozyme or complement, as well as enhanced yeast killing by phagocytes), leading to a better protection against any possible damage (Demers & Bayne, 1997; Ruis & Bayne, 1997). Deleterious e#ects on several humoral and cellular immune parameters have also been observed as a consequence of chronic stress (Yin et al., 1995; Tort et al., 1996b; Bly et al., *Corresponding author. E-mail:
[email protected] 1050–4648/01/020187+11 $35.00/0
187
2001 Academic Press
188
J. ORTUÑO ET AL.
1997; Clearwater & Pankhurst, 1997). Although this general outline of the e#ect of stress on the fish immune system is generally accepted, many points remain to be clarified concerning the e#ects of stress which cannot be accurately defined as belonging to either of the two categories (acute or chronic) previously mentioned. Indeed, intense short term crowding stress, that commonly occurs in several aquacultural practices, seems to possess characteristics of both categories, and its e#ects upon the immune system are poorly understood. In addition, the most important studies on fish stress have usually been carried out by studying immune parameters during stress or immediately after stress (Yin et al., 1995; Tort et al., 1996b; Clearwater & Pankhurst, 1997; Rotllant & Tort, 1997) without establishing when fish recover their normal immune competence, although a knowledge of this recovery would be of great interest for the design of possible preventive strategies. The aim of the present work was to study the influence of intense short-term crowding stress on the gilthead seabream (Sparus aurata L.) innate immune response, as well as to categorise the stress induced by such situations. Special attention was paid to how long stressed fish take to recover their normal immune competence. II. Materials and Methods FISH
Hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.), 120 specimens (15015 g mean weight), obtained from Culmarex S.A. (Murcia, Spain), were kept at 201 C under a natural photoperiod, in four aquaria connected to a running (flow rate 1500 l h 1) sea water (salinity 22‰) system. All the fish were fed 1% body weight per day of commercial dry pellets (ProAqua Nutrición S.A., Palencia, Spain) and allowed to acclimatise to a fish density of 9 kg m 3 for 30 days before the experiment. EXPERIMENTAL PROCEDURE
Fish from the control (undisturbed) group were maintained at a loading of 9 kg m 3. Fish from the experimental group were taken from their aquaria and placed in a well-aerated opaque, covered plastic bucket. Dissolved oxygen was maintained at the same level as in the control group (6·6 mg l 1) using an oxymeter (OXI 197-S) and an aerator (Sirocco). After 2 h of crowding at a fish density of 100 kg m 3 stressed fish were returned to their aquaria and maintained at the control density of 9 kg m 3. Six fish from each group were randomly captured after 0 (immediately after the experiment), 1, 2, 3 and 4 days. In order to maintain the basal fish density throughout the experiment, the water volume in the aquaria was adjusted to the new biomass after each sampling. The experiment was performed twice. SERUM AND LEUCOCYTE ISOLATION
Blood samples of 2 ml were collected from the caudal vein with a 27 gauge needle and a heparinised 1 ml syringe. Aliquots of 1 ml from each blood sample
SHORT-TERM CROWDING STRESS IN SEABREAM
189
were allowed to clot at room temperature overnight. Following centrifugation, the serum was removed and frozen at 80 C until use. Other fresh blood aliquots were resuspended 1:10 in sRPMI-1640 medium [RPMI-1640 medium supplemented with 10 I.U. ml 1 heparin (Sigma), 100 I.U. ml 1 penicillin (Biochrom), 100 g ml 1 streptomycin (Biochrom), 2% foetal calf serum (Gibco) and 0·35% sodium chloride (Sigma) to adjust the medium osmolarity to gilthead seabream plasma osmolarity (353·33 mOs)]. Then, the blood cell suspensions were layered over a 51% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C. After centrifugation, the bands of leucocytes above the Percoll–medium interface were collected with a Pasteur pipette, washed twice, counted and adjusted to 1107 cells ml 1 in sRPMI. Head-kidney was dissected by a ventral incision, cut into small fragments and transferred to 8 ml sRPMI. Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 m). Headkidney cell suspensions were layered over a 34–51% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C. After centrifugation, the bands of leucocytes above the 34–51% interface were collected, washed twice, counted and adjusted to 1107 cells ml 1 in sRPMI. The cell viability of blood and head-kidney leucocytes was greater than 95%, as determined by the trypan blue exclusion test.
GLUCOSE AND CORTISOL LEVELS
Glucose levels in samples of 0·05 ml fresh blood were measured using a commercial kit based on the glucose dehydrogenase enzyme (Boehringer Mannheim Corporation). Serum cortisol levels were quantified by developing a fluorescence polarization immunoassay (TDx/TDxFLx Cortisol assay, Abbott Laboratories, Diagnostics Division).
NATURAL HAEMOLYTIC 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 bu#er (HBSS) containing Mg +2 and EGTA and resuspended at 3% in HBSS. Aliquots of 500 l of test serum (as complement source), diluted in HBSS, were added to 500 l of SRBC 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 C, the samples were centrifuged at 800g for 5 min at 4 C to remove unlysed erythrocytes. The relative haemoglobin content of the supernatants was assessed by measuring their optical density at 540 nm in a spectrophotometer. The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 500 l of distilled water or HBSS to 500 l samples of SRBC, respectively. Heat-inactivated serum was used as a control. The degree of haemolysis (Y) was calculated and the lysis curve for each specimen was obtained by plotting Y (1Y) 1 against the volume of serum added (ml) on a log–log graph. The volume of serum
190
J. ORTUÑO ET AL.
producing 50% haemolysis (ACH50) was determined and the number of ACH50 units ml 1 serum was calculated for each experimental group.
BACTERIA
Vibrio anguillarum strain R82 (serotype 01) (Toranzo & Barja, 1990) was grown in trypticase soy broth (TSB) (Gibco) on agar plates from 1 ml aliquots of stock cultures that had been frozen at 80 C. To label bacteria with fluorescein isothiocyanate (FITC) (Sigma), an isolated colony from each culture was expanded in TSB with FITC (50 or 100 g ml 1) and grown overnight at 20 C in a light protected microenvironment. The optical density of V. anguillarum cell suspensions was measured at 600 nm, the cfu ml 1 being adjusted to 1109 with standard curves of growth in phosphate bu#ered saline solution (PBS). After labelling, free FITC was removed by washing three times in PBS, and the bacteria was heat killed at 60 C for 15 min. After inactivation, the bacteria were washed again. The staining uniformity was examined and then the bacterial cell suspensions were aliquoted and stored at 80 C. PHAGOCYTIC ACTIVITY
The phagocytic activity of gilthead seabream head-kidney leucocytes was studied by flow cytometry according to Esteban et al. (1998). To each 100 l sample of head-kidney leucocyte suspension, aliquots of 10 l FITC-labelled bacteria were added. The samples were then centrifuged (400g, 5 min, 22 C), resuspended and incubated at 22 C for 45 min. At the end of the incubation time the samples were placed on ice to stop phagocytosis and 500 l ice-cold PBS were added to each sample. The fluorescence of the extracellular bacteria (i.e. free bacteria and bacteria adhered to phagocytes but not ingested), was quenched by adding 10 l ice-cold trypan blue (0·4% in PBS) to each sample. Standard samples of FITC-labelled V. anguillarum cells or head-kidney leucocytes were included in each phagocytosis assay. Samples incubated at 4 C were used as negative controls. All samples were analysed in a flow cytometer (Becton Dickinson) with an argon-ion laser adjusted to 488 nm. Instrument settings were adjusted to obtain optimal discrimination of the di#erent cell populations present in seabream head-kidney leucocyte suspensions. Only the population of phagocytes was acquired and analysed in each sample. Analyses were performed upon 10 000 cells that were acquired at a rate of 300 cells s 1. Data were collected in the form of two parameter side scatter (granularity) (SSC) and forward scatter (size) (FSC), or green fluorescence (FL1) and red fluorescence (FL2) dot plots. Fluorescence histograms were obtained on a computerised system, which represented the relative fluorescence on a logarithmic scale. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria (green-FITC fluorescent cells) within the total cell population. The relative number of ingested bacteria per cell (phagocytic capacity) was assessed from the mean fluorescence intensity of the cells.
SHORT-TERM CROWDING STRESS IN SEABREAM
191
RESPIRATORY BURST
The respiratory burst activity of gilthead seabream head-kidney leucocytes was studied by flow cytometry according to Ortun˜ o et al. (2000). Dihydrorhodamine-123 (DHR) (Molecular Probes) was dissolved in N,Ndimethylformamide (Sigma) at a concentration of 1 mM (stock solution), aliquoted and stored at 80 C. Samples (40 l) of head-kidney leucocyte suspensions were mixed with 40 l working DHR solution (10 M in HBSS). After incubation for 5 min at 22 C, 400 l of phorbol 12-myristate 13-acetate (PMA, Sigma) solution (1 ng ml 1 in HBSS) were added to each sample (except control tubes, which received HBSS) and the tubes were incubated for an additional 30 min. Following incubation, the samples were immediately analysed in a flow cytometer as above. Standard samples of leucocytes not incubated with DHR were included in each assay. Samples incubated at 4 C were used as a negative control and samples containing DHR-labelled leucocytes and 30 l 1 mM hydrogen peroxide (Merck) as a positive control. The hydrogen peroxide produced by activated leucocytes specifically oxidise the nonfluorescent DHR to the green fluorescent rhodamine-123. The percentage of activated leucocytes was established as the percentage of green fluorescent cells within the total cell population. The relative quantity of hydrogen peroxide produced was assessed from the mean fluorescence intensity of the cells.
MONOCYTE/MACROPHAGES AND GRANULOCYTES IN HEAD-KIDNEY AND CIRCULATING BLOOD
Head-kidney and blood leucocyte suspensions were analysed in the flow cytometer by acquiring 10 000 cells from each sample, in order to quantify the percentage of monocyte/macrophages and granulocytes among the total cell number, thus permitting any possible change in the total number of leucocytes to be detected. Optimal discrimination of the di#erent cell populations were obtained according to Esteban et al. (1998, 2000). STATISTICAL ANALYSIS
All assays were performed in triplicate and the mean S.E. calculated for each group (n=12). The data from flow cytometric analyses 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 di#erence due to time. Students t-test was used to determine di#erences between groups. Di#erences were considered statistically significant when P
