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Effects of temperature change on the innate cellular and humoral immune responses of orange-spotted grouper Epinephelus coioides and its susceptibility to Vibrio alginolyticus
Fish & Shellfish Immunology 26 (2009) 768–772
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Effects of temperature change on the innate cellular and humoral immune responses of orange-spotted grouper Epinephelus coioides and its susceptibility to Vibrio alginolyticus Ann-Chang Cheng 1, Shao-An Cheng, Yu-Yuan Chen, Jiann-Chu Chen* Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2009 Received in revised form 9 March 2009 Accepted 9 March 2009 Available online 28 March 2009
Orange-spotted grouper Epinephelus coioides held at 27 C were then further cultured at 19, 27 (control), and 35 C, and were examined for innate cellular and humoral responses after 3–96 h. The total leucocyte count, respiratory burst, and phagocytic activity significantly decreased 3, 48, and 96 h after fish were transferred to 19 and 35 C. Both the alternative complement pathway (ACH50) and the lysozyme activity significantly decreased at 3–96 h after fish were transferred to 19 and 35 C. In another experiment, groupers reared at 27 C at 34& salinity were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at a dose of 2.3 109 colony-forming units (cfu) fish1, and then further reared in water temperatures of 19, 27 (control), and 35 C. The cumulative mortalities of V. alginolyticus-injected fish held in 19 and 35 C were significantly higher than that of injected fish held in 27 C. Resistance had decreased after 12 h for the challenged grouper held at 35 C. All injected fish held in 19 C had died after 72 h. It was concluded that at 12 h after transfer of grouper from 27 to 19 and 35 C, immunity was suppressed and resistance against V. alginolyticus had decreased. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Orange-spotted grouper Epinephelus coioides Temperature change Vibrio alginolyticus challenge Innate cellular response Innate humoral response
1. Introduction Groupers, which inhabit subtropical and tropical areas, have become a very popular species of marine teleost currently being cultured in Pacific-rim countries [1]. Among the more than 150 species of grouper worldwide, orange-spotted grouper Epinephelus coioides, malabia grouper Epinephelus malabaricus, brown-marbled grouper Epinephelus fuscoguttatus, giant grouper Epinephelus lanceolatus, and polka dot grouper Cromileptes altivellis are most commonly cultured. Grouper farming has suffered several disease problems like nervous necrosis and sleepy disease [2,3], as well as vibriosis caused by Vibrio alginolyticus and Vibrio carchariae [4–6]. Grouper survive in salinities ranging 11–41&, and in temperatures ranging 21–35 C [7]. Temperature and salinity are the primary environmental parameters, and have been reported to affect growth, survival, physiological function, and immune function in teleosts [8,9]. In fish, the innate immune system can be classified into three factors: 1) physical defense as in the epidermis and mucus,
* Corresponding author. Tel./fax: þ886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 1 Present address: Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung 800, Taiwan, ROC. 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.03.011
2) humoral factors like lysozymes and complement, and 3) cellular factors like phagocytosis and respiratory burst (RB) activity [10]. A change in temperature was reported to affect lysozyme activity in Atlantic halibut [11]. An increase in temperature to >20 C was reported to decrease the susceptibility of sea bass and turbot to hemorrhagic septicemia virus [12]. We assumed that changes in temperature may weaken the immune ability of grouper, and lead to its susceptibility against Vibrio infection. This study was aimed at determining the innate cellular and humoral immune parameters of orange-spotted grouper E. coioides and its resistance to V. alginolyticus when fish were subjected to a temperature change. For the former purpose, total leucocyte count (TLC), RB (release of superoxide anion), phagocytic activity (PA), lysozyme activity and alternative complement were examined.
2. Materials and methods 2.1. Animal Around 500 grouper E. coioides juveniles (3.3 cm, 0.65 0.02 g) were obtained from a private farm in Kaohsiung, Taiwan. They were shipped to our laboratory, and kept in two 7000-l circular tanks with 5000 l of recirculating seawater (34&) that was connected to a biological filtering system (with a 1400-l capacity) at 28–30 C.
A.-C. Cheng et al. / Fish & Shellfish Immunology 26 (2009) 768–772
Water was recirculated at a flow rate of 180 l min1. Fish were fed a commercial diet (Grobest, Taoyuan, Taiwan) to satiation at 8:00 and 20:00 daily until they reached a size of 190 g and were then used for the following experiments. For the susceptibility experiment, there were six treatments (three challenged test groups and three unchallenged control groups). The test and control groups were comprised of 10 fish each kept in triplicate tanks. For the experiment of immune parameter assays, there were 21 treatments (three temperatures of 19, 27, and 35 C combined with seven exposure times of 0, 3, 6, 12, 24, 48 and 96 h). In all tests, fish were fed twice daily with a commercial diet (Grobest) during the experiment. The fish ranged 175–193 g, averaging 190 4.6 g (mean SD) with no significant size difference among treatments. 2.2. Formalin-killed Escherichia coli E. coli (DH5a) was grown overnight in 100 ml tryptic soy broth (TSB) at 37 C. Formaldehyde (37%) was added to give a 2% final concentration and the culture was shaken at 22 C overnight. Stock cultures were centrifuged at 700 g for 10 min at 4 C. The supernatant was removed, and the bacterial pellet was washed twice with 50 ml PBS (phosphate-buffered saline; 8.0 g l1 NaCl, 200 mg l1 KH2PO4, 1.15 g l1 Na2HPO4, 200 mg l1 KCl, 133 mg l1 CaCl2$2H2O and 100 mg l1 MgCl2$6H2O), re-suspended in 50 ml PBS, and kept at 4 C for the phagocytosis test [13]. 2.3. Zymosan Fifty milligrams of zymosan (Z4250, Sigma, St. Louis, MO, USA) in 5 ml PBS was prepared in a capped glass culture tube, and placed in a boiling water bath for 30 min with frequent shaking. The suspension was centrifuged at 600 g for 5 min. The pellet was resuspended in 10 ml chicken serum (C5405, Sigma), incubated for 30 min at 30 C, and then centrifuged at 600 g for 5 min. The supernatant was removed and the zymosan pellet was washed twice with 10 ml PBS, re-suspended in 50 ml PBS to give 1 mg ml1, and stored at 4 C for the respiratory burst assay. 2.4. Culture of V. alginolyticus V. alginolyticus (ATCC17749) obtained from Bioresources Collection and Research Center, Food Industry and Development Institute (Hsinchu, Taiwan) was used for the study. It was cultured on tryptic soy agar (TSA supplemented with 3% NaCl, Difco, Sparks, MD, USA) for 24 h at 28 C before being transferred to 10 ml tryptic soy broth (TSB supplemented with 3% NaCl, Difco), where it remained for 24 h at 28 C as a stock culture for the tests. Broth cultures were centrifuged at 7155 g for 15 min at 4 C. The supernatant was removed and the bacterial pellets were resuspended in saline at 1.2 109 cfu ml1 for the susceptibility test of grouper to V. alginolyticus. The bacterial suspension was obtained based on a standard curve created from a series of different bacterial concentrations and optical densities at 600 nm using a spectrophotometer. 2.5. Effect of water temperature on the susceptibility of E. coioides to V. alginolyticus Challenge tests were conducted in triplicate with 10 fish per replicate. Each fish was injected intraperitoneally with 1000 ml of a V. alginolyticus suspension (1.2 109 cfu ml1) per 100 g of fish weight resulting in 2.3 109 cfu fish1. The fish were then kept in separate 60-l glass aquaria containing 50 l of seawater (10 fish each) at different water temperatures (19, 27, and 35 C). Fish injected
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with an equal volume of tryptic soy broth (TSB) and kept in water with different water temperatures served as the unchallenged control. In total, 180 fish (10 3 6) were used for the study. The mortality of fish in each tank was observed every 12 h for 120 h. The average of the triplicate aquaria was used to express the cumulative mortality. 2.6. Effects of temperature on the immune parameters of E. coioides There were 21 treatments (three temperatures of 19, 27 (control), and 35 C combined with seven exposure times of 0, 3, 6, 12, 24, 48, and 96 h). For the examinations of leucocyte counts, and cellular and humoral parameters, E. coioides (170–190 g) were placed in three replicates of 60-l glass aquaria containing 50 l of seawater (four fish each) at three different temperatures. In total, 252 fish (3 7 4 3) were used for the study. Fish were anaesthetized in 0.02% benzocaine. Blood (1.0–1.5 ml) was individually sampled from the caudal vein using a heparinized syringe (25 g) fitted with a needle. The total leucocyte count was measured using an automated hematology analyzer (KX-21, Sysmex, Tokyo, Japan). The remainder of blood was used for the subsequent tests. Plasma was obtained by centrifugation of a blood sample at 14 700 g (model 5403, Eppendorf, Hamburg, Germany) for 5 min, and used for the lysozyme activity and alternative complement pathway analyses. 2.6.1. Separation of leucocytes Blood (500 ml) was mixed with 500 ml of AL medium (AIM-V medium and Leibovitz’s L 15 medium, GIBCO BRL, Gaithersburg, MD, USA), streptomycin and penicillin. Percoll (55%, P4937, Sigma) was added to the blood and the mixture centrifuged at 400 g (model 5403, Eppendorf) for 15 min at 10 C. Leucocytes were obtained from the interface and washed with AL medium by centrifugation at 600 g for 10 min at 10 C. After centrifugation, leucocytes were suspended in 1 ml AL medium with 5.5 mM glucose. The number of viable cells was analyzed by trypan blue (0.1%) with a haemocytometer [14]. 2.6.2. Measurement of the innate cellular response Respiratory burst of leucocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O 2 ) production [15]. Briefly, 100 ml of a leucocyte suspension in AL medium was placed in a microplate (96 wells) in triplicate tubes. One hundred microlitres of the NBT solution (0.1% in Hank’s balanced salt solution) and 100 ml zymosan was added, allowed to react for 30 min at room temperature, then 100 ml of 100% methanol was added to stop the reaction, and the mixture was centrifuged. One minute later, the mixed solution was discarded, and the microplates were washed three times with 100 ml of 70% methanol and air-dried. One hundred and twenty microlitres of 2 M KOH and 140 ml of dimethyl sulfoxide (DMSO) were added to dissolve the insoluble formazan crystals formed from the reduction of NBT. The absorbance at 630 nm was measured spectrometrically in triplicates with a microplate reader (model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RB is expressed as NBT reduction in 100 ml of leucocyte suspension. Phagocytosis was studied based on a previously described method [16]. Briefly 300 ml of leucocyte suspensions in AL medium was added to triplicate tubes. Three hundred microlitres of formalin-killed E. coli in PBS was added to each tube and incubated at room temperature for 1 h. Then, 900 ml of cold PBS was added, and the tubes were centrifuged at 300 g for 5 min at 4 C. The supernatants were discarded and the pellets were taken up and smeared on glass slides. The slides were air-dried, then stained with Giemsa solution (Sigma). Cells were counted using
A.-C. Cheng et al. / Fish & Shellfish Immunology 26 (2009) 768–772
a microscope (Olympus PM30, Tokyo, Japan). The percentage of leucocytes ingesting E. coli (A) and the number of E. coli ingested per phagocyte (B) were calculated by enumerating 100 leucocytes under a microscope. The phagocytic activity was expressed as the phagocytic index (PI) (PI ¼ A B) [17,18]. 2.6.3. Measurement of the innate humoral response Lysozyme activity was measured based on a turbidimetric assay [19]. Briefly, a standard suspension (0.2 mg ml1) of Micrococcus lysodeikticus (Sigma) was prepared in 0.05 M sodium phosphate buffer (pH 6.2). Test plasma (10 ml) was added to 200 ml of the bacterial suspension in a 96-well microplate, and the decreases in absorbance at 520 nm were recorded after 1 and 4 min at 22 C. A standard solution containing 0, 10, 20, 30, 50, and 100 units ml1 of hen egg white lysozyme (L6876, Sigma) was used to construct a standard curve. A unit of lysozyme activity was defined as the amount of plasma causing a reduction in absorbance of 0.001 min1. Activity of the alternative complement pathway was assayed using sheep red blood cells (SRBCs, R3378, Sigma) as targets [20]. Briefly, SRBCs were washed three times in phenol red-free Hank’s balanced salt solution (HBSS) supplemented with 0.5 mM Mg2þ and 10 mM EGTA, and re-suspended at 3% (v/v) in HBSS. The plasma sample which was diluted (1:50) to different volumes ranging from 0.1 to 0.25 ml was dispensed into a series of test tubes, the total volume was made up to 0.25 ml with the same buffer, and this was then added to 0.1 ml of the SRBC suspension. After incubation for 1 h at 25 C, the mixture was centrifuged at 400 g for 5 min at 4 C. The relative hemoglobin content of the supernatant was assessed by measuring the optical density (OD) at 414 nm using a spectrophotometer (model U-2000 Hitachi, Tokyo, Japan). 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 in the test tubes without SRBC were also included 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/(1 Y) against the volume of plasma added (ml) on a log10–log10 scaled graph. The volume of plasma yielding 50% haemolysis (ACH50) was determined, and the number of ACH50 units ml1 was obtained for each experimental group. 2.7. Statistical analysis Tukey’s multiple-range tests were used to determine the significant differences among treatment groups using SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (susceptibility test) were normalized using an arcsine transformation before the analysis. Differences were considered statistically significant when p < 0.05. 3. Results 3.1. Effects of a temperature change on the susceptibility of grouper to V. alginolyticus All unchallenged control groupers survived. By contrast, onset of mortality occurred earliest for the challenged grouper held at 19 C (12 h), next for the challenged grouper held at 35 C (24 h), and then for the challenged grouper held at 27 C (72 h). From 24 to 120 h post-infection, the cumulative mortalities for grouper held at 19 and 35 C were significantly higher than that of fish held at 27 C. The cumulative mortality of the challenged group held at 27 C was the lowest, and the highest was for the challenged fish held at 19 C (Fig. 1).
3.2. Effects of temperature on the innate immune parameters of grouper TLCs significantly decreased by 18% and 30% for fish 3 and 96 h after being transferred to 19 C, respectively. TLCs significantly decreased by 15% and 58% for fish 3 and 96 h after being transferred to 35 C, respectively (Fig. 2A). RB significantly decreased by 16% and 28% for fish 3 and 96 h after being transferred to 19 C, respectively. RB significantly decreased by 26% and 18% for fish 3 and 96 h after being transferred to 35 C, respectively (Fig. 2B). PA significantly decreased by 14%, 10%, and 40% for fish 3, 6, and 96 h after being transferred to 19 C, respectively. PA significantly decreased by 16%, 14%, and 21% for fish 3, 6, and 96 h after being transferred to 35 C, respectively (Fig. 2C). Both the ACH50 and the lysozyme activity significantly decreased for fish at 3–96 h after being transferred to 19 and 35 C (Fig. 3).
4. Discussion PA of tilapia Oreochromis niloticus significantly decreased when transferred from 25 to 12 C [21]. Leucocyte counts, RB, and PA were significantly higher for tilapia reared at 27 C, but significantly decreased in fish 24 h after being transferred to a low temperature (19 C) and high temperature (35 C) [13]. Similarly, these parameters were significantly higher for grouper reared at 27 C, but significantly decreased when fish were transferred to 19 and 35 C. Therefore, transfer to a low and high temperature causes decreases in leucocyte counts and cellular responses. Both serum complement activity and lysozyme activities of gilthead sea bream Sparus aurata sampled in winter temperature period (11 C) was significantly lower, as compared to those of fish sampled during warm periods (15–21 C) [22]. Several studies have been conducted on the innate humoral response of fish subjected to temperature stresses. For example, both serum complement and lysozyme activities of sea bream S. aurata significantly decreased after the decrease of temperature from 18 to 11 C at a rate of 1 C per day [23]. Both lysozyme activity and ACH50 significantly decreased in tilapia 12–96 h after being transferred from 27 to 19 C [13]. It is generally accepted that lower temperature adversely affects specific immune responses mediated by T helper cells [24]. In the present study, the lower levels of ACH50 and lysozyme activity of the grouper well matched the resistance against V. alginolyticus, indicating decreased immunity of grouper subjected to a low temperature (19 C), and a high temperature (35 C).
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Fig. 3. Mean (SE) ACH50 (A), and lysozyme activity (B) of orange-spotted grouper Epinephelus coioides kept at 27 C at the beginning, and then 3, 6, 12, 24, 48, and 96 h after being transferred to 19, 27, and 35 C. See Fig. 1 for statistical information.
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Time elapsed (h) Fig. 2. Mean (SE) total leucocyte count (A), respiratory bursts (B), and phagocytic activity (C) of orange-spotted grouper Epinephelus coioides kept at 27 C at the beginning, and then 3, 6, 12, 24, 48, and 96 h after being transferred to 19, 27, and 35 C. See Fig. 1 for statistical information.
It is known that the primary response of fish to stress like crowding and handling is the production of catecholamine and cortisol by the interrenal gland [25–29]. Crowding induces an increase in cortisol and depression of serum agglutinating activity of sea bream S. aurata [30]. Cortisol administration causes a decrease in the resistance of fish against fungal and bacterial pathogens [31,32], decreases the number of phagocytes, and causes a depressive effect on phagocytosis [29]. Both RB and PA decreased when leucocytes of sea bream S. aurata were incubated with cortisol [33]. The fact that grouper E. coioides when transferred from 27 to 19 and 35 C show decreased innate cellular responses suggests that the resulting increase in cortisol may lead to suppression of immunity, and depression of resistance against V. alginolyticus. In conclusion, the present study documented that E. coioides reared at 27 C, and subjected to a temperature change to 19 C or 35 C showed decreases in the leucocyte count, and cellular and humoral responses, together with increased susceptibility to
This research was supported partially by a grant from the Center for the Marine Bioscience and Biotechnology, National Taiwan Ocean University. We thank Mr. S.T. Yeh for his assistance with the experiments.
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Effects of temperature change on the innate cellular and humoral immune responses of orange-spotted grouper Epinephelus coioides and its susceptibility to Vibrio alginolyticus Ann-Chang Cheng 1, Shao-An Cheng, Yu-Yuan Chen, Jiann-Chu Chen* Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 February 2009 Received in revised form 9 March 2009 Accepted 9 March 2009 Available online 28 March 2009
Orange-spotted grouper Epinephelus coioides held at 27 C were then further cultured at 19, 27 (control), and 35 C, and were examined for innate cellular and humoral responses after 3–96 h. The total leucocyte count, respiratory burst, and phagocytic activity significantly decreased 3, 48, and 96 h after fish were transferred to 19 and 35 C. Both the alternative complement pathway (ACH50) and the lysozyme activity significantly decreased at 3–96 h after fish were transferred to 19 and 35 C. In another experiment, groupers reared at 27 C at 34& salinity were injected with Vibrio alginolyticus grown in tryptic soy broth (TSB) at a dose of 2.3 109 colony-forming units (cfu) fish1, and then further reared in water temperatures of 19, 27 (control), and 35 C. The cumulative mortalities of V. alginolyticus-injected fish held in 19 and 35 C were significantly higher than that of injected fish held in 27 C. Resistance had decreased after 12 h for the challenged grouper held at 35 C. All injected fish held in 19 C had died after 72 h. It was concluded that at 12 h after transfer of grouper from 27 to 19 and 35 C, immunity was suppressed and resistance against V. alginolyticus had decreased. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Orange-spotted grouper Epinephelus coioides Temperature change Vibrio alginolyticus challenge Innate cellular response Innate humoral response
1. Introduction Groupers, which inhabit subtropical and tropical areas, have become a very popular species of marine teleost currently being cultured in Pacific-rim countries [1]. Among the more than 150 species of grouper worldwide, orange-spotted grouper Epinephelus coioides, malabia grouper Epinephelus malabaricus, brown-marbled grouper Epinephelus fuscoguttatus, giant grouper Epinephelus lanceolatus, and polka dot grouper Cromileptes altivellis are most commonly cultured. Grouper farming has suffered several disease problems like nervous necrosis and sleepy disease [2,3], as well as vibriosis caused by Vibrio alginolyticus and Vibrio carchariae [4–6]. Grouper survive in salinities ranging 11–41&, and in temperatures ranging 21–35 C [7]. Temperature and salinity are the primary environmental parameters, and have been reported to affect growth, survival, physiological function, and immune function in teleosts [8,9]. In fish, the innate immune system can be classified into three factors: 1) physical defense as in the epidermis and mucus,
* Corresponding author. Tel./fax: þ886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 1 Present address: Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung 800, Taiwan, ROC. 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.03.011
2) humoral factors like lysozymes and complement, and 3) cellular factors like phagocytosis and respiratory burst (RB) activity [10]. A change in temperature was reported to affect lysozyme activity in Atlantic halibut [11]. An increase in temperature to >20 C was reported to decrease the susceptibility of sea bass and turbot to hemorrhagic septicemia virus [12]. We assumed that changes in temperature may weaken the immune ability of grouper, and lead to its susceptibility against Vibrio infection. This study was aimed at determining the innate cellular and humoral immune parameters of orange-spotted grouper E. coioides and its resistance to V. alginolyticus when fish were subjected to a temperature change. For the former purpose, total leucocyte count (TLC), RB (release of superoxide anion), phagocytic activity (PA), lysozyme activity and alternative complement were examined.
2. Materials and methods 2.1. Animal Around 500 grouper E. coioides juveniles (3.3 cm, 0.65 0.02 g) were obtained from a private farm in Kaohsiung, Taiwan. They were shipped to our laboratory, and kept in two 7000-l circular tanks with 5000 l of recirculating seawater (34&) that was connected to a biological filtering system (with a 1400-l capacity) at 28–30 C.
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Water was recirculated at a flow rate of 180 l min1. Fish were fed a commercial diet (Grobest, Taoyuan, Taiwan) to satiation at 8:00 and 20:00 daily until they reached a size of 190 g and were then used for the following experiments. For the susceptibility experiment, there were six treatments (three challenged test groups and three unchallenged control groups). The test and control groups were comprised of 10 fish each kept in triplicate tanks. For the experiment of immune parameter assays, there were 21 treatments (three temperatures of 19, 27, and 35 C combined with seven exposure times of 0, 3, 6, 12, 24, 48 and 96 h). In all tests, fish were fed twice daily with a commercial diet (Grobest) during the experiment. The fish ranged 175–193 g, averaging 190 4.6 g (mean SD) with no significant size difference among treatments. 2.2. Formalin-killed Escherichia coli E. coli (DH5a) was grown overnight in 100 ml tryptic soy broth (TSB) at 37 C. Formaldehyde (37%) was added to give a 2% final concentration and the culture was shaken at 22 C overnight. Stock cultures were centrifuged at 700 g for 10 min at 4 C. The supernatant was removed, and the bacterial pellet was washed twice with 50 ml PBS (phosphate-buffered saline; 8.0 g l1 NaCl, 200 mg l1 KH2PO4, 1.15 g l1 Na2HPO4, 200 mg l1 KCl, 133 mg l1 CaCl2$2H2O and 100 mg l1 MgCl2$6H2O), re-suspended in 50 ml PBS, and kept at 4 C for the phagocytosis test [13]. 2.3. Zymosan Fifty milligrams of zymosan (Z4250, Sigma, St. Louis, MO, USA) in 5 ml PBS was prepared in a capped glass culture tube, and placed in a boiling water bath for 30 min with frequent shaking. The suspension was centrifuged at 600 g for 5 min. The pellet was resuspended in 10 ml chicken serum (C5405, Sigma), incubated for 30 min at 30 C, and then centrifuged at 600 g for 5 min. The supernatant was removed and the zymosan pellet was washed twice with 10 ml PBS, re-suspended in 50 ml PBS to give 1 mg ml1, and stored at 4 C for the respiratory burst assay. 2.4. Culture of V. alginolyticus V. alginolyticus (ATCC17749) obtained from Bioresources Collection and Research Center, Food Industry and Development Institute (Hsinchu, Taiwan) was used for the study. It was cultured on tryptic soy agar (TSA supplemented with 3% NaCl, Difco, Sparks, MD, USA) for 24 h at 28 C before being transferred to 10 ml tryptic soy broth (TSB supplemented with 3% NaCl, Difco), where it remained for 24 h at 28 C as a stock culture for the tests. Broth cultures were centrifuged at 7155 g for 15 min at 4 C. The supernatant was removed and the bacterial pellets were resuspended in saline at 1.2 109 cfu ml1 for the susceptibility test of grouper to V. alginolyticus. The bacterial suspension was obtained based on a standard curve created from a series of different bacterial concentrations and optical densities at 600 nm using a spectrophotometer. 2.5. Effect of water temperature on the susceptibility of E. coioides to V. alginolyticus Challenge tests were conducted in triplicate with 10 fish per replicate. Each fish was injected intraperitoneally with 1000 ml of a V. alginolyticus suspension (1.2 109 cfu ml1) per 100 g of fish weight resulting in 2.3 109 cfu fish1. The fish were then kept in separate 60-l glass aquaria containing 50 l of seawater (10 fish each) at different water temperatures (19, 27, and 35 C). Fish injected
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with an equal volume of tryptic soy broth (TSB) and kept in water with different water temperatures served as the unchallenged control. In total, 180 fish (10 3 6) were used for the study. The mortality of fish in each tank was observed every 12 h for 120 h. The average of the triplicate aquaria was used to express the cumulative mortality. 2.6. Effects of temperature on the immune parameters of E. coioides There were 21 treatments (three temperatures of 19, 27 (control), and 35 C combined with seven exposure times of 0, 3, 6, 12, 24, 48, and 96 h). For the examinations of leucocyte counts, and cellular and humoral parameters, E. coioides (170–190 g) were placed in three replicates of 60-l glass aquaria containing 50 l of seawater (four fish each) at three different temperatures. In total, 252 fish (3 7 4 3) were used for the study. Fish were anaesthetized in 0.02% benzocaine. Blood (1.0–1.5 ml) was individually sampled from the caudal vein using a heparinized syringe (25 g) fitted with a needle. The total leucocyte count was measured using an automated hematology analyzer (KX-21, Sysmex, Tokyo, Japan). The remainder of blood was used for the subsequent tests. Plasma was obtained by centrifugation of a blood sample at 14 700 g (model 5403, Eppendorf, Hamburg, Germany) for 5 min, and used for the lysozyme activity and alternative complement pathway analyses. 2.6.1. Separation of leucocytes Blood (500 ml) was mixed with 500 ml of AL medium (AIM-V medium and Leibovitz’s L 15 medium, GIBCO BRL, Gaithersburg, MD, USA), streptomycin and penicillin. Percoll (55%, P4937, Sigma) was added to the blood and the mixture centrifuged at 400 g (model 5403, Eppendorf) for 15 min at 10 C. Leucocytes were obtained from the interface and washed with AL medium by centrifugation at 600 g for 10 min at 10 C. After centrifugation, leucocytes were suspended in 1 ml AL medium with 5.5 mM glucose. The number of viable cells was analyzed by trypan blue (0.1%) with a haemocytometer [14]. 2.6.2. Measurement of the innate cellular response Respiratory burst of leucocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O 2 ) production [15]. Briefly, 100 ml of a leucocyte suspension in AL medium was placed in a microplate (96 wells) in triplicate tubes. One hundred microlitres of the NBT solution (0.1% in Hank’s balanced salt solution) and 100 ml zymosan was added, allowed to react for 30 min at room temperature, then 100 ml of 100% methanol was added to stop the reaction, and the mixture was centrifuged. One minute later, the mixed solution was discarded, and the microplates were washed three times with 100 ml of 70% methanol and air-dried. One hundred and twenty microlitres of 2 M KOH and 140 ml of dimethyl sulfoxide (DMSO) were added to dissolve the insoluble formazan crystals formed from the reduction of NBT. The absorbance at 630 nm was measured spectrometrically in triplicates with a microplate reader (model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RB is expressed as NBT reduction in 100 ml of leucocyte suspension. Phagocytosis was studied based on a previously described method [16]. Briefly 300 ml of leucocyte suspensions in AL medium was added to triplicate tubes. Three hundred microlitres of formalin-killed E. coli in PBS was added to each tube and incubated at room temperature for 1 h. Then, 900 ml of cold PBS was added, and the tubes were centrifuged at 300 g for 5 min at 4 C. The supernatants were discarded and the pellets were taken up and smeared on glass slides. The slides were air-dried, then stained with Giemsa solution (Sigma). Cells were counted using
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a microscope (Olympus PM30, Tokyo, Japan). The percentage of leucocytes ingesting E. coli (A) and the number of E. coli ingested per phagocyte (B) were calculated by enumerating 100 leucocytes under a microscope. The phagocytic activity was expressed as the phagocytic index (PI) (PI ¼ A B) [17,18]. 2.6.3. Measurement of the innate humoral response Lysozyme activity was measured based on a turbidimetric assay [19]. Briefly, a standard suspension (0.2 mg ml1) of Micrococcus lysodeikticus (Sigma) was prepared in 0.05 M sodium phosphate buffer (pH 6.2). Test plasma (10 ml) was added to 200 ml of the bacterial suspension in a 96-well microplate, and the decreases in absorbance at 520 nm were recorded after 1 and 4 min at 22 C. A standard solution containing 0, 10, 20, 30, 50, and 100 units ml1 of hen egg white lysozyme (L6876, Sigma) was used to construct a standard curve. A unit of lysozyme activity was defined as the amount of plasma causing a reduction in absorbance of 0.001 min1. Activity of the alternative complement pathway was assayed using sheep red blood cells (SRBCs, R3378, Sigma) as targets [20]. Briefly, SRBCs were washed three times in phenol red-free Hank’s balanced salt solution (HBSS) supplemented with 0.5 mM Mg2þ and 10 mM EGTA, and re-suspended at 3% (v/v) in HBSS. The plasma sample which was diluted (1:50) to different volumes ranging from 0.1 to 0.25 ml was dispensed into a series of test tubes, the total volume was made up to 0.25 ml with the same buffer, and this was then added to 0.1 ml of the SRBC suspension. After incubation for 1 h at 25 C, the mixture was centrifuged at 400 g for 5 min at 4 C. The relative hemoglobin content of the supernatant was assessed by measuring the optical density (OD) at 414 nm using a spectrophotometer (model U-2000 Hitachi, Tokyo, Japan). 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 in the test tubes without SRBC were also included 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/(1 Y) against the volume of plasma added (ml) on a log10–log10 scaled graph. The volume of plasma yielding 50% haemolysis (ACH50) was determined, and the number of ACH50 units ml1 was obtained for each experimental group. 2.7. Statistical analysis Tukey’s multiple-range tests were used to determine the significant differences among treatment groups using SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (susceptibility test) were normalized using an arcsine transformation before the analysis. Differences were considered statistically significant when p < 0.05. 3. Results 3.1. Effects of a temperature change on the susceptibility of grouper to V. alginolyticus All unchallenged control groupers survived. By contrast, onset of mortality occurred earliest for the challenged grouper held at 19 C (12 h), next for the challenged grouper held at 35 C (24 h), and then for the challenged grouper held at 27 C (72 h). From 24 to 120 h post-infection, the cumulative mortalities for grouper held at 19 and 35 C were significantly higher than that of fish held at 27 C. The cumulative mortality of the challenged group held at 27 C was the lowest, and the highest was for the challenged fish held at 19 C (Fig. 1).
3.2. Effects of temperature on the innate immune parameters of grouper TLCs significantly decreased by 18% and 30% for fish 3 and 96 h after being transferred to 19 C, respectively. TLCs significantly decreased by 15% and 58% for fish 3 and 96 h after being transferred to 35 C, respectively (Fig. 2A). RB significantly decreased by 16% and 28% for fish 3 and 96 h after being transferred to 19 C, respectively. RB significantly decreased by 26% and 18% for fish 3 and 96 h after being transferred to 35 C, respectively (Fig. 2B). PA significantly decreased by 14%, 10%, and 40% for fish 3, 6, and 96 h after being transferred to 19 C, respectively. PA significantly decreased by 16%, 14%, and 21% for fish 3, 6, and 96 h after being transferred to 35 C, respectively (Fig. 2C). Both the ACH50 and the lysozyme activity significantly decreased for fish at 3–96 h after being transferred to 19 and 35 C (Fig. 3).
4. Discussion PA of tilapia Oreochromis niloticus significantly decreased when transferred from 25 to 12 C [21]. Leucocyte counts, RB, and PA were significantly higher for tilapia reared at 27 C, but significantly decreased in fish 24 h after being transferred to a low temperature (19 C) and high temperature (35 C) [13]. Similarly, these parameters were significantly higher for grouper reared at 27 C, but significantly decreased when fish were transferred to 19 and 35 C. Therefore, transfer to a low and high temperature causes decreases in leucocyte counts and cellular responses. Both serum complement activity and lysozyme activities of gilthead sea bream Sparus aurata sampled in winter temperature period (11 C) was significantly lower, as compared to those of fish sampled during warm periods (15–21 C) [22]. Several studies have been conducted on the innate humoral response of fish subjected to temperature stresses. For example, both serum complement and lysozyme activities of sea bream S. aurata significantly decreased after the decrease of temperature from 18 to 11 C at a rate of 1 C per day [23]. Both lysozyme activity and ACH50 significantly decreased in tilapia 12–96 h after being transferred from 27 to 19 C [13]. It is generally accepted that lower temperature adversely affects specific immune responses mediated by T helper cells [24]. In the present study, the lower levels of ACH50 and lysozyme activity of the grouper well matched the resistance against V. alginolyticus, indicating decreased immunity of grouper subjected to a low temperature (19 C), and a high temperature (35 C).
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Fig. 3. Mean (SE) ACH50 (A), and lysozyme activity (B) of orange-spotted grouper Epinephelus coioides kept at 27 C at the beginning, and then 3, 6, 12, 24, 48, and 96 h after being transferred to 19, 27, and 35 C. See Fig. 1 for statistical information.
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Acknowledgements
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Time elapsed (h) Fig. 2. Mean (SE) total leucocyte count (A), respiratory bursts (B), and phagocytic activity (C) of orange-spotted grouper Epinephelus coioides kept at 27 C at the beginning, and then 3, 6, 12, 24, 48, and 96 h after being transferred to 19, 27, and 35 C. See Fig. 1 for statistical information.
It is known that the primary response of fish to stress like crowding and handling is the production of catecholamine and cortisol by the interrenal gland [25–29]. Crowding induces an increase in cortisol and depression of serum agglutinating activity of sea bream S. aurata [30]. Cortisol administration causes a decrease in the resistance of fish against fungal and bacterial pathogens [31,32], decreases the number of phagocytes, and causes a depressive effect on phagocytosis [29]. Both RB and PA decreased when leucocytes of sea bream S. aurata were incubated with cortisol [33]. The fact that grouper E. coioides when transferred from 27 to 19 and 35 C show decreased innate cellular responses suggests that the resulting increase in cortisol may lead to suppression of immunity, and depression of resistance against V. alginolyticus. In conclusion, the present study documented that E. coioides reared at 27 C, and subjected to a temperature change to 19 C or 35 C showed decreases in the leucocyte count, and cellular and humoral responses, together with increased susceptibility to
This research was supported partially by a grant from the Center for the Marine Bioscience and Biotechnology, National Taiwan Ocean University. We thank Mr. S.T. Yeh for his assistance with the experiments.
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