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Immune condition of Chlamys farreri in response to acute temperature challenge
Aquaculture 271 (2007) 479 – 487 www.elsevier.com/locate/aqua-online
Immune condition of Chlamys farreri in response to acute temperature challenge Muyan Chen a,b , Hongsheng Yang a,⁎, Maryse Delaporte c , Sanjun Zhao a,b a
c
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Graduate University, Chinese Academy of Sciences, Beijing 100049, China Department of Pathology and Microbiology, Atlantic Veterinary College, Charlottetown, C1E4P3, Prince Edward Island, Canada Received 9 March 2007; received in revised form 20 April 2007; accepted 20 April 2007
Abstract The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 °C to 11 °C, 23 °C and 28 °C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 °C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 °C to 28 °C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 °C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. © 2007 Elsevier B.V. All rights reserved. Keywords: Acute temperature challenge; Chlamys farreri; Biomarkers; Haemocyte activities; High tolerance
1. Introduction Culture of the Zhikong scallop Chlamys farreri (Jones and Presten) substantially contributes to the northern Chinese aquaculture industry. However, aquaculture of this species has been thwarted by summer mortality for several decades causing extensive economic losses (Zhang and Yang, 1999a,b; Xiao et al., 2005). Such mortality outbreaks have been reported for the Pacific oyster Crassostrea gigas (Glude, 1975; ⁎ Corresponding author. E-mail address: [email protected] (H. Yang). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.051
Hershberger et al., 1984; Goulletquer et al., 1998) and the Eastern oyster Crassostrea virginica (Shumway, 1996). Until now, causes of mortalities remain unclear, but it is generally admitted that water temperature contribute as one of the most important environmental factors involved in scallops summer mortality by affecting scallop physiology (reproduction, energetic metabolism, growth and immunity) (Zhang and Yang, 1999b; Jiang, 2004). The internal defense system of bivalves consists of both humoral and cellular immunity. Haemocytes, which are the most important cells involved in defense, circulate in the whole body within the haemolymph (Cheng, 1981).
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Fig. 1. Jiaozhou Bay, ⁎ showing location of sampling site.
Since bivalves are both osmo- and thermo-conformers, haemolymph reflects salinity and temperature of external environmental conditions in which bivalves are exposed (Shumway, 1977). As a consequence, haemocytes immune parameters have been used as biomarkers to assess environmental condition effects on mollusc health. In this way, total haemocyte count and phagocytic activity of haemocytes have been demonstrated to be susceptible to environmental temperature variations in the European flat oyster Ostrea edulis (Fisher et al., 1987), C. virginica
(Fisher et al., 1989), the Taiwan abalone Haliotis diversicolor supertexta (Cheng et al., 2004), C. gigas (Gagnaire et al., 2006), and Chamelea gallina (Monari et al., 2007). As for example, total haemocyte count of O. edulis and H. diversicolor supertexta increased while the percentage of phagocytic haemocytes decreased when animals were exposed to acute temperature elevation (Fisher et al., 1987; Cheng et al., 2004). Cheng et al. (2004) also reported an increase of reactive oxygen species (ROS) production, involved in pathogen
Fig. 2. Effects of 72 h of acute temperature challenge on total haemocyte counts of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
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Fig. 3. Effects of 72 h of acute temperature challenge on the percentage of phagocytic haemocytes of C. farreri (expressed as percentage of haemocytes that have engulfed three beads and more). Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
degradation, in H. diversicolor supertexta during acute thermal stress (elevation of seawater temperature from 28 °C to 32 °C). The extent of ROS damage on cells (i.e. membrane damage, DNA breakage, enzyme inhibition and amino acid oxidation) depends on the effectiveness of the antioxidant defense (Michiels and Remacle, 1988), especially superoxide dismutase (SOD) activity which is the first and the most important enzyme involved in ROS detoxification (Downs et al., 2001). Unsurprisingly, SOD activity in clam C. gallina has been demonstrated to depend on temperature (Monari et al., 2007). Thus, in the study of these authors, a continuous decrease in haemocyte Mn-SOD and Cu–Zn-SOD activities was observed in haemocyte lysate with temperature elevation.
Meanwhile, in cell-free haemolymph, the highest MnSOD activity was recorded at 30 °C. Besides cellular functions, acid phosphatase (ACP), a typical lysosomal enzyme involve in killing and digesting microbial pathogens in bivalves, also appears to be sensitive to temperature variation (Liu et al., 2004). A significant increase of ACP activity was recorded in haemolymph of C. farreri maintained at 30 °C for two weeks (Liu et al., 2004). On the other hand, Camus et al. (2000) indicated that lysosomal membranes of the Blue mussels Mytilus edulis challenged at 0 °C were destabilized compared to mussels held at 10 °C. It is suggested that low temperature may affect the stability of lysosomal and haemocyte membrane and as a consequence trigger the
Fig. 4. Effects of 72 h of acute temperature challenge on the reactive oxygen species production by haemocytes of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
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Fig. 5. Effects of 72 h of acute temperature challenge on ACP activity, in cell-free haemolymph and haemocyte lysate of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
hypersynthesis of ACP, which is subsequently released into the haemolymph (Suresh and Mohandas, 1990). Also, several studies have reported changes in haemocyte activities of mollusc species depending on seawater temperature variations (seasonal, extreme and short-term variation) associated or not with pathogenic infections, as for example for the soft shell clam Mya arenaria (Abele et al., 2002), C. virginica (Hégaret et al., 2003b), the Manila clam Ruditapes philippinarum (Paillard et al., 2004), C. gigas (Gagnaire et al., 2006), and the Venus clam C. gallina (Monari et al., 2007). Such information is lacking for the scallop C. farreri. However, a better understanding of the relationship between the defense mechanisms of scallops and environmental conditions is necessary for the development of ecological adjustments and disease management strategies for scallop C. farreri aquaculture. The aim of the present study was to assess the effect of acute temperature variations (suddenly transferred from 17 °C to 11 °C, 23 °C, 28 °C for 72 h) on several immune conditions of haemocyte in C. farreri (total haemocyte counts, percentage of phagocytic haemocyte, ROS
production, SOD and ACP activities in cell-free haemolymph and haemocyte lysate). 2. Materials and methods 2.1. Scallops conditioning Scallops, C. farreri (3.5 ± 0.32 cm of shell height), were collected from Jiaozhou Bay (Fig. 1) (in March, 2006) and acclimatized at 17 °C for two weeks before stress application. Scallops were maintained in lantern nets suspended in 800-l tanks containing aerated filtered seawater (31 ppt) renewed daily. Scallops were daily fed superfluous microalgae Phaeodactylum tricornutum Bohlin. 2.2. Exposure to experimental temperatures After acclimation at 17 °C, scallops were randomly transferred in tanks at 11 °C, 23 °C and 28 °C for a period of 72 h. Three tank replicates were set up for each
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Fig. 6. Effects of 72 h of acute temperature challenge on SOD activity, in cell-free haemolymph and haemocyte lysate of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
experimental temperature. After 1 h, 24 h and 72 h of stress application, 5 scallops per replicate were sampled. Scallops acclimated at 17 °C were sampled as controls. During the acclimation period as well the stress exposure, no relevant variations in seawater pH or salinity values were recorded. 2.3. Haemolymph collection Haemolymph was withdrawn from the anterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily in individual microcentrifuge tubes maintained on ice to retard cell clumping. For each experimental temperature (11, 23 and 28 °C) and each sampling point (0, 1, 24, and 72 h), three pools of 5 scallops were analyzed. 2.4. Measurements of haemocyte parameters by flow cytometer Haemocyte parameters of C. farreri were monitored using a FACSVantage (BD Biosciences) flow cytometer. As recommended by flow cytometer manufacturer,
samples were filtered through a 50-μm mesh to eliminate potential large debris which might block the flow cytometer. Methods to measure haemocyte parameters are described hereafter. 2.4.1. Haemolymph preparation An aliquot of haemolymph was mixed with anticoagulant solution (1:1; Glucose 20.8 g l− 1, EDTA 20 mM, Sodium chloride 20 g l− 1, Tris–HCl 0.05 M, pH = 7.4) according to Richard et al. (1997) in order to prevent haemocyte clotting and was used as haemolymph working solution. 2.4.2. Total haemocyte count 200 μl of the initial pooled haemolymph (not working solution) was fixed with 200 μl of 6% formalin solution in sterile filtered seawater (FSSW). Then, haemocytes were incubated with SYBR Green I (10%), a nucleic acid specific dye, in darkness. After 30 min of incubation at 18 °C, SYBR Green fluorescence of the cells was detected at 500–530 nm by flow cytometer on the FL1 detector. This protocol was adapted from Hégaret et al. (2003a).
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2.4.3. Phagocytosis assay 200 μl of haemolymph working solution was centrifuged at 780 g for 10 min. Pelleted haemocytes were resuspended in FSSW. Thereafter, 30 μl of fluorescent beads (Fluoresbrite YG Microspheres, 2.00 μm (Polysciences), at a final concentration of 0.3% of the commercial suspension) was added to each tube. After 60 min of incubation at 18 °C, haemocytes were fixed with 230 μl of 6% formalin solution in FSSW prior to analysis. The phagocytic activity of haemocytes was estimated as the percentage of haemocytes that had engulfed three beads or more. This protocol was adapted from the study of Delaporte et al. (2003).
analysis with the SPSS 11.5 statistical software. When a significant effect (pb 0.05) of duration and temperature was found, a multiple comparison (Tukey) test was conducted to compare the significant difference among treatments. Percentage data were transformed (arcsin of the square root) before ANOVA, but presented in figures as nontransformed percentage.
2.4.4. Reactive oxygen species production The reactive oxygen species (ROS) production of haemocytes was measured following an adapted method of Lambert et al. (2003) for C. gigas and using 2′7′dichlorofluorescein diacetate (DCFH-DA). 400 μl of haemolymph working solution was transferred into a tube maintained on ice. 4 μl of DCFH-DA (final concentration of 0.01 mM) was added and tubes were incubated at 18 °C. After 1 h of incubation, DCF fluorescence, quantitatively related to the ROS production of haemocytes without any stimulation, was measured at 500–530 nm by flow cytometer. DCF fluorescence was expressed in arbitrary units (AU).
3.1. Total haemocyte count
2.5. ACP and SOD activities assay
3. Results Only 10% mortality was found during treatments for 72 h at 28 °C, and no mortalities were observed for those maintained at 11 °C and 23 °C.
Total haemocyte count (THC) was significantly affected by the temperature and duration of the stress (2-way ANOVA, p b 0.001). After 1 h of acute temperature stress, THC significantly increased from 3.7 × 107 cells ml− 1 initially to an average of 5.3 × 107 cells ml− 1 in scallops transferred to 11, 23 and 28 °C (Fig. 2, one-way ANOVA, p b 0.001, p b 0.01 and p b 0.05 respectively). At the end of the 72−h stress application, THC of scallops placed at 11 °C remained significantly higher with 5.2 × 107 cells ml− 1 than those of other treatment groups, for which THC have returned roughly to the initial values with an average of 4.0 × 107 cells ml− 1.
ACP and SOD activities were quantified in both cellfree haemolymph and haemocyte lysate. Initial subsample of pooled haemolymph was centrifuged at 780 g for 10 min. The supernatant, corresponding to cell-free haemolymph, was collected, whereas the haemocyte pellet was treated according to Sun and Li (2000). Briefly, pelleted haemocytes were resuspended in the same volume of distilled water for lysis, and then centrifuged at 12,000 g for 15 min to obtain haemocyte lysate. Cellfree haemolymph and haemocyte lysate were frozen and stored at −80 °C until analysis. ACP and SOD activities were measured according to the methods of Song (1991) and Deng et al. (1991), respectively by disodium phenyl orthophosphate method and by pyrogallol self-oxidation. Results of ACP and SOD measurement in cell-free haemolymph and haemocyte lysate were expressed as specific activity, i.e. U 100 ml− 1 and U ml− 1 respectively.
3.2. Percentage of phagocytic haemocytes
2.6. Statistic analysis
3.3. Reactive oxygen species production
Two-way analysis of variance (2-way ANOVA) was performed for all the haemocyte immune parameters
ROS production was significantly affected by the temperature (2-way ANOVA, p b 0.001), but not by
Percentage of phagocytic haemocytes was significantly affected by the temperature (2-way ANOVA, p b 0.001), but not by the duration of the stress application (2-way ANOVA, p N 0.05). Percentage of phagocytic haemocytes of scallops challenged at 28 °C was significantly lower with respect to those placed at 11 °C and 23 °C after 1h stress application (Fig. 3, one-way ANOVA, p b 0.01 and p b 0.05) and significantly decreased over the whole stress application, from 13.9% initially to 6.3% at the end of the experiment (Fig. 3, one-way ANOVA, p b 0.001). Phagocytic activity of scallops challenged at 11 °C and 23 °C remained comparatively stable over the experiment (average of 13.7% and 12.7 respectively).
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the duration of the stress application (2-way ANOVA, p N 0.05). A significant decrease of ROS production was observed for scallops transferred to 23 °C after 1 h of acute temperature stress (Fig. 4, one-way ANOVA, p b 0.01), and after 72 h of stress application for scallops transferred to 11 °C (Fig. 4, one-way ANOVA, p b 0.05). By contrast, ROS production of haemocytes from scallops challenged at 28 °C was significantly higher than initial values during the whole stress application (Fig. 4, one-way ANOVA, p b 0.01, p b 0.01 and p b 0.05 respectively). 3.4. Acid phosphatase activity (ACP) ACP activity was significantly affected by the temperature and stress duration both in cell-free haemolymph (CFH) and haemocyte lysate (HL) (2way ANOVA, p b 0.01 and p b 0.05 respectively). A significant decrease of ACP activity in CFH was observed for scallops challenged at 28 °C (Fig. 5A, oneway ANOVA, p b 0.001). ACP activity in CFH decreased from 0.98 U 100 ml− 1 to 0.11 U 100 ml− 1 after 24 h of the stress application and down to 0.16 U 100 ml− 1 at the end of the stress application. ACP activity in CFH of scallops challenged at 23 °C decreased down to 0.2 U 100 ml− 1, but only at the end of the 72 h of stress application. By contrast, ACP activity of scallops in CFH challenged at 11 °C remained quite stable and similar to initial values (average of 1.2 U ml− 1, p N 0.05). In HL, ACP activity significantly increased from 0.46 U 100 ml− 1 to 1.3 U 100 ml− 1 for scallops challenged at 28 °C after 1 h of acute temperature challenge, then decreased back to initial value at the end of the stress application (Fig. 5B). Conversely, a significant decrease of ACP activity was observed in HL from scallops challenged at 11 °C after 1 h and 24 h of temperature challenge (Fig. 5B, one-way ANOVA, p b 0.05 and p b 0.01). However, ACP activity in HL of those scallops was significantly higher at the end of the 72 h of stress application than initially (Fig. 5B, one-way ANOVA, p b 0.01). 3.5. Superoxide dismutase activity (SOD) No significant effect of temperature variation and stress duration was observed on SOD activity in CFH (Fig. 6A, 2-way ANOVA, p N 0.05). However, SOD activity in HL was significantly affected by those parameters (2-way ANOVA, p b 0.05). After 1 h of acute temperature challenge, SOD activity in HL of scallops challenged at 23 °C and 28 °C significantly increased (Fig. 6B, one-way ANOVA, p b 0.05 and p b 0.001 respectively) from 35.2 U ml− 1 to
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an average of 66.9 U ml− 1. While SOD activity in HL of scallops exposed to 28 °C decreased back to 30.5 U ml− 1 at the end of the stress application, a significant increase of SOD activity from 35.2 U ml− 1 initially to 57.9 U ml− 1 was observed in HL of scallops challenged at 11 °C (Fig. 6B, one-way ANOVA, p b 0.05). 4. Discussion Although seawater in northern China, where C. farreri is cultured widely, has submitted to high (important) thermal variation (even to 29 °C) and that temperature is known to be one of the most important natural factor affecting the defense mechanisms in bivalves (Abele et al., 2002; Hégaret et al., 2003a,b; Liu et al., 2004; Paillard et al., 2004; Gagnaire et al., 2006; Monari et al., 2007), up to date, no study has been performed to assess the haemocyte activity changes of C. farreri to acute temperature variations. In the present study, changes in total haemocyte counts (THC) demonstrated that C. farreri is sensitive to acute temperature challenge. THC significantly increased after 1 h of acute thermal stress whatever the temperature of the stress application (11 °C, 23 °C or 28 °C), which indicated that acute variations of temperature could temporarily affect the ability of mollusc haemocytes to resist foreign invasion (Fisher et al., 1987). Also, it is suggested that increased THC in scallop challenged at 23 °C and 28 °C due to acute temperature elevation may result in cell proliferation or cell migration from tissues into the circulation as proposed for H. diversicolor supertexta by Cheng et al. (2004), for C. gigas by Gagnaire et al. (2006), and for C. gallina by Monari et al. (2007). In addition, under low temperature stress, the increase of THC observed in scallop C. farreri challenged at 11 °C was also in agreement with Cheng et al. (2004). The authors indicated that transferring H. diversicolor supertexta from 28 °C to 20 °C induced an increase of total haemocyte counts. However, many other studies reported a decrease in THC in crustacean due to a decrease or increase in water temperature (Truscott and White, 1990; VargasAlbores et al., 1998; Le Moullac and Haffner, 2000). It is supposed that differences in species, difference in haemocyte sub-population proportions (i.e. hyalinocyte vs granulocyte), experimental settings (seasonal pattern), duration and severity of the stress application have affected the outcome of the experiments. With regard to haemocyte functions, the percentage of phagocytic haemocytes decreased significantly for the scallops challenged at 28 °C, which was not correlated with changes in THC. Similar observations were reported in H. diversicolor supertexta (Cheng et al., 2004) and C. gallina
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(Monari et al., 2007). But, as proposed by Alvarez et al. (1989) and Chu and La Peyre (1993), it can be also suggested that temperatures above a certain threshold may result in stress condition for haemocytes. As a consequence, haemocytes are less active to phagocyte (pathogens or beads) although the total haemocyte counts increased. Temperature challenge also affects lysosomal enzyme in C. farreri. In the present study, a significant decrease in ACP activity was observed in CFH for scallops challenged at 28 °C. This is consistent with the decrease of phagocytosis activity reported for those scallops since the release of the lysosomal enzyme into serum (i.e. CFH) depends on haemocyte degranulation during the phagocytosis process (Pipe, 1990). Moreover, variation of ACP activity in HL of scallops challenged at 11 °C may be due to the destabilization of lysosomal membrane induced by the rapid temperature decrease from 17 °C to 11 °C during the first 24 h of stress application (Zhang et al., 2006). Then, lysosomal membranes may become stable again due to C. farreri ability to tolerate low temperature. Meanwhile, the significant increase of ACP activity observed in HL of scallops challenged at 28 °C may reflect the optimal temperature for ACP activity in haemocytes. Moreover, it has been suggested that bivalve molluscs produce ROS production and activate antioxidative enzymes only in response to acute changes in temperature, especially heat shock stress (Abele et al., 2002). In the present study, a transitory increase of SOD activity in haemocyte lysate of scallops challenged at 23 °C and 28 °C for 1 h was detected concomitantly with an increase of ROS production; however, it seems that SOD was no longer active to depress the oxidative damage of haemocytes of scallops over the whole stress application even though SOD is the first and the most important defense line in the antioxidant system (Downs et al., 2001). Interestingly, no significant effect of sudden temperature elevation was observed on the total SOD activity (Cu–Zn and Mn) in CFH. This result support the statement of Sun and Li (2000) on the fact that Cu–Zn-SOD activity was very stable even under extremely high temperature (80 °C) and was only detected in CFH of C. farreri (Sun and Li, 2000). In conclusion, the homeostatic capabilities appear to be compromised for C. farreri transferred from 17 °C to 28 °C. It is supposed that the overall health of the scallop would be compromised and the likelihood of being affected by pathogens might increase. By contrast, scallops possessed a high tolerance to a rapid decrease of seawater temperature in the present study. However, scallops response to temperature in laboratory experi-
ments may be influenced by environmental and endogenous conditions previously experienced by animals in the field. To support such a hypothesis, it is important to highlight that compared with the mass mortality of scallops sampled in summer, which naturally was more stressed due to experiencing high temperature and reproductive effort (Yuan et al., 2000), the reduced mortality during treatments for 72 h at 28 °C (10%) for scallops collected in March was observed in our present study. Similarly, Monari et al. (2007) found that in C. gallina, mortality rates were significantly lower collected in March (28%) compared with the animals collected in early autumn (48%) during treatments for 7 days at 30 °C. Moreover, it is reported that mass mortality usually occurs as the scallops are entering their second year from late July into August (Xiao et al., 2005); in our study, one year old scallops were used. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 30671614), the National Key Foundational Research Project of China (No. 2007CB407305) and the Hi-tech Research and Development Program of China (No. 2006AA100304/ 2006AA100307). References Abele, D., Heise, K., Pörtner, H.O., Puntarulo, S., 2002. Temperaturedependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria. J. Exp. Biol. 205, 1831–1841. Alvarez, M.R., Friedl, F.E., Johnson, J., Hinsch, G.W., 1989. Factors affecting in vitro phagocytosis by oyster hemocytes. J. Invertebr. Pathol. 54, 233–241. Camus, L., Grøsvik, B.E., Børseth, J.F., Jones, M.B., Depledge, M.H., 2000. Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures. Mar. Environ. Res. 50, 325–329. Cheng, T.C., 1981. Bivalves. In: Ratcliffe, N.A., Rowley, A.F. (Eds.), Invertebrate Blood Cells I. Academic Press, London, pp. 233–299. Cheng, W., Hsiao, I.S., Hsu, C.H., Chen, J.C., 2004. Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Fish Shellfish Immunol. 17, 235–243. Chu, F.E., La Peyre, J.F., 1993. Development of disease caused by the parasite, Perkinsus marinus and defense-related hemolymph factors in three populations of oysters from Chesapeake Bay, USA. J. Shellfish Res. 12, 21–27. Delaporte, M., Soudant, P., Moal, J., Lambert, C., Quéré, C., Miner, P., Choquet, G., Paillard, C., Samain, J.F., 2003. Effect of a monospecific algal diet on immune functions in two bivalves species Crassostrea gigas and Ruditapes philippinarum. J. Exp. Biol. 206, 3053–3064.
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Monari, M., Matozzo, V., Foschi, J., Cattani, O., Serrazanetti, G.P., Marin, M.G., 2007. Effects of high temperature on functional responses of haemocytes in the clam Chamelea gallina. Fish Shellfish Immunol. 22, 1–17. Paillard, C., Allam, B., Oubella, R., 2004. Effect of temperature on defence parameters in Manila clam Ruditapes philippinarum challenged with Vibrio tapetis. Dis. Aquat. Org. 59, 249–262. Pipe, R.K., 1990. Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis. Histochem. J. 22, 595–603. Richard, K.P., Sophia, R.F., Jackie, A.C., 1997. The separation and characterization of haemocytes from the mussel Mytilus edulis. Cell Tissue Res. 289, 537–545. Shumway, S.E., 1977. Effect of salinity fluctuation on the osmotic pressure and Na+, Ca2+ and Mg2+ ion concentrations in the hemolymph of bivalve molluscs. Mar. Biol. 41, 153–177. Shumway, S.E., 1996. Natural environmental factors. In: Kennedy, V.S., Newell, R.I.E., Eble, A.F. (Eds.), The Eastern Oyster Crassostrea virginica. Maryland Sea Grant, College Park, MD, USA, pp. 467–513. Song, S.J., 1991. The Clinician's Handbook. Shanghai Scientific and Technical Publishers. (185 pp. in Chinese). Sun, H.S., Li, G.Y., 2000. Activities and properties of superoxide dismutase and catalase in the haemolymph of Chlamys farreri. Oceanol. Limnol. Sin. 3, 259–265 (in Chinese with English abstract). Suresh, K., Mohandas, A., 1990. Hemolymph acid phosphatase activity pattern in copper-stressed bivalves. J. Invertebr. Pathol. 55, 118–125. Truscott, R., White, K.N., 1990. The influence of metal and temperature stress on the immune system of crabs. Funct. Ecol. 4, 455–461. Vargas-Albores, F., Baltazar, P.H., Clark, G.P., Barajas, F.M., 1998. Influence of temperature and salinity on the yellowleg shrimp, Penaeus californieneis Holmes, prophenoloxidase system. Aquac. Res. 29, 549–553. Xiao, J., Ford, S.E., Yang, H.S., Zhang, G.F., Zhang, F.S., Guo, X.M., 2005. Studies on mass summer mortality of cultured Zhikong scallops (Chlamys farreri Jones et Preston) in China. Aquaculture 250, 605–615. Yuan, Y.X., Qu, K.M., Chen, J.F., Guo, F., Li, Q.F., Cui, Y., 2000. Adaptability of Chlamys farreri to environment-effects of temperature on survival, respiration, ingestion and digestion. J. Fish. Sci. China. 3, 24–27 (in Chinese with English abstract). Zhang, F.S., Yang, H.S., 1999a. Strategic and counter measures to resolve mass mortality problems of Chlamys farreri. Mar. Sci. 2, 38–42 (in Chinese with English abstract). Zhang, F.S., Yang, H.S., 1999b. Analysis of the causes of mass mortality of farming Chlamys farreri in summer in coastal areas of Shandong, China. Mar. Sci. 1, 44–47 (in Chinese with English abstract). Zhang, Z.H., Li, X.X., Vandepeer, M., Zhao, W., 2006. Effects of water temperature and air exposure on the lysosomal membrane stability of hemocytes in pacific oysters, Crassostrea gigas (Thunberg). Aquaculture 256, 502–509.
Immune condition of Chlamys farreri in response to acute temperature challenge Muyan Chen a,b , Hongsheng Yang a,⁎, Maryse Delaporte c , Sanjun Zhao a,b a
c
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Graduate University, Chinese Academy of Sciences, Beijing 100049, China Department of Pathology and Microbiology, Atlantic Veterinary College, Charlottetown, C1E4P3, Prince Edward Island, Canada Received 9 March 2007; received in revised form 20 April 2007; accepted 20 April 2007
Abstract The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 °C to 11 °C, 23 °C and 28 °C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 °C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 °C to 28 °C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 °C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. © 2007 Elsevier B.V. All rights reserved. Keywords: Acute temperature challenge; Chlamys farreri; Biomarkers; Haemocyte activities; High tolerance
1. Introduction Culture of the Zhikong scallop Chlamys farreri (Jones and Presten) substantially contributes to the northern Chinese aquaculture industry. However, aquaculture of this species has been thwarted by summer mortality for several decades causing extensive economic losses (Zhang and Yang, 1999a,b; Xiao et al., 2005). Such mortality outbreaks have been reported for the Pacific oyster Crassostrea gigas (Glude, 1975; ⁎ Corresponding author. E-mail address: [email protected] (H. Yang). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.051
Hershberger et al., 1984; Goulletquer et al., 1998) and the Eastern oyster Crassostrea virginica (Shumway, 1996). Until now, causes of mortalities remain unclear, but it is generally admitted that water temperature contribute as one of the most important environmental factors involved in scallops summer mortality by affecting scallop physiology (reproduction, energetic metabolism, growth and immunity) (Zhang and Yang, 1999b; Jiang, 2004). The internal defense system of bivalves consists of both humoral and cellular immunity. Haemocytes, which are the most important cells involved in defense, circulate in the whole body within the haemolymph (Cheng, 1981).
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Fig. 1. Jiaozhou Bay, ⁎ showing location of sampling site.
Since bivalves are both osmo- and thermo-conformers, haemolymph reflects salinity and temperature of external environmental conditions in which bivalves are exposed (Shumway, 1977). As a consequence, haemocytes immune parameters have been used as biomarkers to assess environmental condition effects on mollusc health. In this way, total haemocyte count and phagocytic activity of haemocytes have been demonstrated to be susceptible to environmental temperature variations in the European flat oyster Ostrea edulis (Fisher et al., 1987), C. virginica
(Fisher et al., 1989), the Taiwan abalone Haliotis diversicolor supertexta (Cheng et al., 2004), C. gigas (Gagnaire et al., 2006), and Chamelea gallina (Monari et al., 2007). As for example, total haemocyte count of O. edulis and H. diversicolor supertexta increased while the percentage of phagocytic haemocytes decreased when animals were exposed to acute temperature elevation (Fisher et al., 1987; Cheng et al., 2004). Cheng et al. (2004) also reported an increase of reactive oxygen species (ROS) production, involved in pathogen
Fig. 2. Effects of 72 h of acute temperature challenge on total haemocyte counts of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
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Fig. 3. Effects of 72 h of acute temperature challenge on the percentage of phagocytic haemocytes of C. farreri (expressed as percentage of haemocytes that have engulfed three beads and more). Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
degradation, in H. diversicolor supertexta during acute thermal stress (elevation of seawater temperature from 28 °C to 32 °C). The extent of ROS damage on cells (i.e. membrane damage, DNA breakage, enzyme inhibition and amino acid oxidation) depends on the effectiveness of the antioxidant defense (Michiels and Remacle, 1988), especially superoxide dismutase (SOD) activity which is the first and the most important enzyme involved in ROS detoxification (Downs et al., 2001). Unsurprisingly, SOD activity in clam C. gallina has been demonstrated to depend on temperature (Monari et al., 2007). Thus, in the study of these authors, a continuous decrease in haemocyte Mn-SOD and Cu–Zn-SOD activities was observed in haemocyte lysate with temperature elevation.
Meanwhile, in cell-free haemolymph, the highest MnSOD activity was recorded at 30 °C. Besides cellular functions, acid phosphatase (ACP), a typical lysosomal enzyme involve in killing and digesting microbial pathogens in bivalves, also appears to be sensitive to temperature variation (Liu et al., 2004). A significant increase of ACP activity was recorded in haemolymph of C. farreri maintained at 30 °C for two weeks (Liu et al., 2004). On the other hand, Camus et al. (2000) indicated that lysosomal membranes of the Blue mussels Mytilus edulis challenged at 0 °C were destabilized compared to mussels held at 10 °C. It is suggested that low temperature may affect the stability of lysosomal and haemocyte membrane and as a consequence trigger the
Fig. 4. Effects of 72 h of acute temperature challenge on the reactive oxygen species production by haemocytes of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
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Fig. 5. Effects of 72 h of acute temperature challenge on ACP activity, in cell-free haemolymph and haemocyte lysate of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
hypersynthesis of ACP, which is subsequently released into the haemolymph (Suresh and Mohandas, 1990). Also, several studies have reported changes in haemocyte activities of mollusc species depending on seawater temperature variations (seasonal, extreme and short-term variation) associated or not with pathogenic infections, as for example for the soft shell clam Mya arenaria (Abele et al., 2002), C. virginica (Hégaret et al., 2003b), the Manila clam Ruditapes philippinarum (Paillard et al., 2004), C. gigas (Gagnaire et al., 2006), and the Venus clam C. gallina (Monari et al., 2007). Such information is lacking for the scallop C. farreri. However, a better understanding of the relationship between the defense mechanisms of scallops and environmental conditions is necessary for the development of ecological adjustments and disease management strategies for scallop C. farreri aquaculture. The aim of the present study was to assess the effect of acute temperature variations (suddenly transferred from 17 °C to 11 °C, 23 °C, 28 °C for 72 h) on several immune conditions of haemocyte in C. farreri (total haemocyte counts, percentage of phagocytic haemocyte, ROS
production, SOD and ACP activities in cell-free haemolymph and haemocyte lysate). 2. Materials and methods 2.1. Scallops conditioning Scallops, C. farreri (3.5 ± 0.32 cm of shell height), were collected from Jiaozhou Bay (Fig. 1) (in March, 2006) and acclimatized at 17 °C for two weeks before stress application. Scallops were maintained in lantern nets suspended in 800-l tanks containing aerated filtered seawater (31 ppt) renewed daily. Scallops were daily fed superfluous microalgae Phaeodactylum tricornutum Bohlin. 2.2. Exposure to experimental temperatures After acclimation at 17 °C, scallops were randomly transferred in tanks at 11 °C, 23 °C and 28 °C for a period of 72 h. Three tank replicates were set up for each
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Fig. 6. Effects of 72 h of acute temperature challenge on SOD activity, in cell-free haemolymph and haemocyte lysate of C. farreri. Letters indicate significant differences among treatments (Mean ± S.D; n = 3 pools, p b 0.05). Asterisks indicate significant differences with respect to initial values (⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001).
experimental temperature. After 1 h, 24 h and 72 h of stress application, 5 scallops per replicate were sampled. Scallops acclimated at 17 °C were sampled as controls. During the acclimation period as well the stress exposure, no relevant variations in seawater pH or salinity values were recorded. 2.3. Haemolymph collection Haemolymph was withdrawn from the anterior adductor muscle with a 1-ml plastic syringe fitted with a 25-gauge needle and stored temporarily in individual microcentrifuge tubes maintained on ice to retard cell clumping. For each experimental temperature (11, 23 and 28 °C) and each sampling point (0, 1, 24, and 72 h), three pools of 5 scallops were analyzed. 2.4. Measurements of haemocyte parameters by flow cytometer Haemocyte parameters of C. farreri were monitored using a FACSVantage (BD Biosciences) flow cytometer. As recommended by flow cytometer manufacturer,
samples were filtered through a 50-μm mesh to eliminate potential large debris which might block the flow cytometer. Methods to measure haemocyte parameters are described hereafter. 2.4.1. Haemolymph preparation An aliquot of haemolymph was mixed with anticoagulant solution (1:1; Glucose 20.8 g l− 1, EDTA 20 mM, Sodium chloride 20 g l− 1, Tris–HCl 0.05 M, pH = 7.4) according to Richard et al. (1997) in order to prevent haemocyte clotting and was used as haemolymph working solution. 2.4.2. Total haemocyte count 200 μl of the initial pooled haemolymph (not working solution) was fixed with 200 μl of 6% formalin solution in sterile filtered seawater (FSSW). Then, haemocytes were incubated with SYBR Green I (10%), a nucleic acid specific dye, in darkness. After 30 min of incubation at 18 °C, SYBR Green fluorescence of the cells was detected at 500–530 nm by flow cytometer on the FL1 detector. This protocol was adapted from Hégaret et al. (2003a).
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2.4.3. Phagocytosis assay 200 μl of haemolymph working solution was centrifuged at 780 g for 10 min. Pelleted haemocytes were resuspended in FSSW. Thereafter, 30 μl of fluorescent beads (Fluoresbrite YG Microspheres, 2.00 μm (Polysciences), at a final concentration of 0.3% of the commercial suspension) was added to each tube. After 60 min of incubation at 18 °C, haemocytes were fixed with 230 μl of 6% formalin solution in FSSW prior to analysis. The phagocytic activity of haemocytes was estimated as the percentage of haemocytes that had engulfed three beads or more. This protocol was adapted from the study of Delaporte et al. (2003).
analysis with the SPSS 11.5 statistical software. When a significant effect (pb 0.05) of duration and temperature was found, a multiple comparison (Tukey) test was conducted to compare the significant difference among treatments. Percentage data were transformed (arcsin of the square root) before ANOVA, but presented in figures as nontransformed percentage.
2.4.4. Reactive oxygen species production The reactive oxygen species (ROS) production of haemocytes was measured following an adapted method of Lambert et al. (2003) for C. gigas and using 2′7′dichlorofluorescein diacetate (DCFH-DA). 400 μl of haemolymph working solution was transferred into a tube maintained on ice. 4 μl of DCFH-DA (final concentration of 0.01 mM) was added and tubes were incubated at 18 °C. After 1 h of incubation, DCF fluorescence, quantitatively related to the ROS production of haemocytes without any stimulation, was measured at 500–530 nm by flow cytometer. DCF fluorescence was expressed in arbitrary units (AU).
3.1. Total haemocyte count
2.5. ACP and SOD activities assay
3. Results Only 10% mortality was found during treatments for 72 h at 28 °C, and no mortalities were observed for those maintained at 11 °C and 23 °C.
Total haemocyte count (THC) was significantly affected by the temperature and duration of the stress (2-way ANOVA, p b 0.001). After 1 h of acute temperature stress, THC significantly increased from 3.7 × 107 cells ml− 1 initially to an average of 5.3 × 107 cells ml− 1 in scallops transferred to 11, 23 and 28 °C (Fig. 2, one-way ANOVA, p b 0.001, p b 0.01 and p b 0.05 respectively). At the end of the 72−h stress application, THC of scallops placed at 11 °C remained significantly higher with 5.2 × 107 cells ml− 1 than those of other treatment groups, for which THC have returned roughly to the initial values with an average of 4.0 × 107 cells ml− 1.
ACP and SOD activities were quantified in both cellfree haemolymph and haemocyte lysate. Initial subsample of pooled haemolymph was centrifuged at 780 g for 10 min. The supernatant, corresponding to cell-free haemolymph, was collected, whereas the haemocyte pellet was treated according to Sun and Li (2000). Briefly, pelleted haemocytes were resuspended in the same volume of distilled water for lysis, and then centrifuged at 12,000 g for 15 min to obtain haemocyte lysate. Cellfree haemolymph and haemocyte lysate were frozen and stored at −80 °C until analysis. ACP and SOD activities were measured according to the methods of Song (1991) and Deng et al. (1991), respectively by disodium phenyl orthophosphate method and by pyrogallol self-oxidation. Results of ACP and SOD measurement in cell-free haemolymph and haemocyte lysate were expressed as specific activity, i.e. U 100 ml− 1 and U ml− 1 respectively.
3.2. Percentage of phagocytic haemocytes
2.6. Statistic analysis
3.3. Reactive oxygen species production
Two-way analysis of variance (2-way ANOVA) was performed for all the haemocyte immune parameters
ROS production was significantly affected by the temperature (2-way ANOVA, p b 0.001), but not by
Percentage of phagocytic haemocytes was significantly affected by the temperature (2-way ANOVA, p b 0.001), but not by the duration of the stress application (2-way ANOVA, p N 0.05). Percentage of phagocytic haemocytes of scallops challenged at 28 °C was significantly lower with respect to those placed at 11 °C and 23 °C after 1h stress application (Fig. 3, one-way ANOVA, p b 0.01 and p b 0.05) and significantly decreased over the whole stress application, from 13.9% initially to 6.3% at the end of the experiment (Fig. 3, one-way ANOVA, p b 0.001). Phagocytic activity of scallops challenged at 11 °C and 23 °C remained comparatively stable over the experiment (average of 13.7% and 12.7 respectively).
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the duration of the stress application (2-way ANOVA, p N 0.05). A significant decrease of ROS production was observed for scallops transferred to 23 °C after 1 h of acute temperature stress (Fig. 4, one-way ANOVA, p b 0.01), and after 72 h of stress application for scallops transferred to 11 °C (Fig. 4, one-way ANOVA, p b 0.05). By contrast, ROS production of haemocytes from scallops challenged at 28 °C was significantly higher than initial values during the whole stress application (Fig. 4, one-way ANOVA, p b 0.01, p b 0.01 and p b 0.05 respectively). 3.4. Acid phosphatase activity (ACP) ACP activity was significantly affected by the temperature and stress duration both in cell-free haemolymph (CFH) and haemocyte lysate (HL) (2way ANOVA, p b 0.01 and p b 0.05 respectively). A significant decrease of ACP activity in CFH was observed for scallops challenged at 28 °C (Fig. 5A, oneway ANOVA, p b 0.001). ACP activity in CFH decreased from 0.98 U 100 ml− 1 to 0.11 U 100 ml− 1 after 24 h of the stress application and down to 0.16 U 100 ml− 1 at the end of the stress application. ACP activity in CFH of scallops challenged at 23 °C decreased down to 0.2 U 100 ml− 1, but only at the end of the 72 h of stress application. By contrast, ACP activity of scallops in CFH challenged at 11 °C remained quite stable and similar to initial values (average of 1.2 U ml− 1, p N 0.05). In HL, ACP activity significantly increased from 0.46 U 100 ml− 1 to 1.3 U 100 ml− 1 for scallops challenged at 28 °C after 1 h of acute temperature challenge, then decreased back to initial value at the end of the stress application (Fig. 5B). Conversely, a significant decrease of ACP activity was observed in HL from scallops challenged at 11 °C after 1 h and 24 h of temperature challenge (Fig. 5B, one-way ANOVA, p b 0.05 and p b 0.01). However, ACP activity in HL of those scallops was significantly higher at the end of the 72 h of stress application than initially (Fig. 5B, one-way ANOVA, p b 0.01). 3.5. Superoxide dismutase activity (SOD) No significant effect of temperature variation and stress duration was observed on SOD activity in CFH (Fig. 6A, 2-way ANOVA, p N 0.05). However, SOD activity in HL was significantly affected by those parameters (2-way ANOVA, p b 0.05). After 1 h of acute temperature challenge, SOD activity in HL of scallops challenged at 23 °C and 28 °C significantly increased (Fig. 6B, one-way ANOVA, p b 0.05 and p b 0.001 respectively) from 35.2 U ml− 1 to
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an average of 66.9 U ml− 1. While SOD activity in HL of scallops exposed to 28 °C decreased back to 30.5 U ml− 1 at the end of the stress application, a significant increase of SOD activity from 35.2 U ml− 1 initially to 57.9 U ml− 1 was observed in HL of scallops challenged at 11 °C (Fig. 6B, one-way ANOVA, p b 0.05). 4. Discussion Although seawater in northern China, where C. farreri is cultured widely, has submitted to high (important) thermal variation (even to 29 °C) and that temperature is known to be one of the most important natural factor affecting the defense mechanisms in bivalves (Abele et al., 2002; Hégaret et al., 2003a,b; Liu et al., 2004; Paillard et al., 2004; Gagnaire et al., 2006; Monari et al., 2007), up to date, no study has been performed to assess the haemocyte activity changes of C. farreri to acute temperature variations. In the present study, changes in total haemocyte counts (THC) demonstrated that C. farreri is sensitive to acute temperature challenge. THC significantly increased after 1 h of acute thermal stress whatever the temperature of the stress application (11 °C, 23 °C or 28 °C), which indicated that acute variations of temperature could temporarily affect the ability of mollusc haemocytes to resist foreign invasion (Fisher et al., 1987). Also, it is suggested that increased THC in scallop challenged at 23 °C and 28 °C due to acute temperature elevation may result in cell proliferation or cell migration from tissues into the circulation as proposed for H. diversicolor supertexta by Cheng et al. (2004), for C. gigas by Gagnaire et al. (2006), and for C. gallina by Monari et al. (2007). In addition, under low temperature stress, the increase of THC observed in scallop C. farreri challenged at 11 °C was also in agreement with Cheng et al. (2004). The authors indicated that transferring H. diversicolor supertexta from 28 °C to 20 °C induced an increase of total haemocyte counts. However, many other studies reported a decrease in THC in crustacean due to a decrease or increase in water temperature (Truscott and White, 1990; VargasAlbores et al., 1998; Le Moullac and Haffner, 2000). It is supposed that differences in species, difference in haemocyte sub-population proportions (i.e. hyalinocyte vs granulocyte), experimental settings (seasonal pattern), duration and severity of the stress application have affected the outcome of the experiments. With regard to haemocyte functions, the percentage of phagocytic haemocytes decreased significantly for the scallops challenged at 28 °C, which was not correlated with changes in THC. Similar observations were reported in H. diversicolor supertexta (Cheng et al., 2004) and C. gallina
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(Monari et al., 2007). But, as proposed by Alvarez et al. (1989) and Chu and La Peyre (1993), it can be also suggested that temperatures above a certain threshold may result in stress condition for haemocytes. As a consequence, haemocytes are less active to phagocyte (pathogens or beads) although the total haemocyte counts increased. Temperature challenge also affects lysosomal enzyme in C. farreri. In the present study, a significant decrease in ACP activity was observed in CFH for scallops challenged at 28 °C. This is consistent with the decrease of phagocytosis activity reported for those scallops since the release of the lysosomal enzyme into serum (i.e. CFH) depends on haemocyte degranulation during the phagocytosis process (Pipe, 1990). Moreover, variation of ACP activity in HL of scallops challenged at 11 °C may be due to the destabilization of lysosomal membrane induced by the rapid temperature decrease from 17 °C to 11 °C during the first 24 h of stress application (Zhang et al., 2006). Then, lysosomal membranes may become stable again due to C. farreri ability to tolerate low temperature. Meanwhile, the significant increase of ACP activity observed in HL of scallops challenged at 28 °C may reflect the optimal temperature for ACP activity in haemocytes. Moreover, it has been suggested that bivalve molluscs produce ROS production and activate antioxidative enzymes only in response to acute changes in temperature, especially heat shock stress (Abele et al., 2002). In the present study, a transitory increase of SOD activity in haemocyte lysate of scallops challenged at 23 °C and 28 °C for 1 h was detected concomitantly with an increase of ROS production; however, it seems that SOD was no longer active to depress the oxidative damage of haemocytes of scallops over the whole stress application even though SOD is the first and the most important defense line in the antioxidant system (Downs et al., 2001). Interestingly, no significant effect of sudden temperature elevation was observed on the total SOD activity (Cu–Zn and Mn) in CFH. This result support the statement of Sun and Li (2000) on the fact that Cu–Zn-SOD activity was very stable even under extremely high temperature (80 °C) and was only detected in CFH of C. farreri (Sun and Li, 2000). In conclusion, the homeostatic capabilities appear to be compromised for C. farreri transferred from 17 °C to 28 °C. It is supposed that the overall health of the scallop would be compromised and the likelihood of being affected by pathogens might increase. By contrast, scallops possessed a high tolerance to a rapid decrease of seawater temperature in the present study. However, scallops response to temperature in laboratory experi-
ments may be influenced by environmental and endogenous conditions previously experienced by animals in the field. To support such a hypothesis, it is important to highlight that compared with the mass mortality of scallops sampled in summer, which naturally was more stressed due to experiencing high temperature and reproductive effort (Yuan et al., 2000), the reduced mortality during treatments for 72 h at 28 °C (10%) for scallops collected in March was observed in our present study. Similarly, Monari et al. (2007) found that in C. gallina, mortality rates were significantly lower collected in March (28%) compared with the animals collected in early autumn (48%) during treatments for 7 days at 30 °C. Moreover, it is reported that mass mortality usually occurs as the scallops are entering their second year from late July into August (Xiao et al., 2005); in our study, one year old scallops were used. Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 30671614), the National Key Foundational Research Project of China (No. 2007CB407305) and the Hi-tech Research and Development Program of China (No. 2006AA100304/ 2006AA100307). References Abele, D., Heise, K., Pörtner, H.O., Puntarulo, S., 2002. Temperaturedependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria. J. Exp. Biol. 205, 1831–1841. Alvarez, M.R., Friedl, F.E., Johnson, J., Hinsch, G.W., 1989. Factors affecting in vitro phagocytosis by oyster hemocytes. J. Invertebr. Pathol. 54, 233–241. Camus, L., Grøsvik, B.E., Børseth, J.F., Jones, M.B., Depledge, M.H., 2000. Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures. Mar. Environ. Res. 50, 325–329. Cheng, T.C., 1981. Bivalves. In: Ratcliffe, N.A., Rowley, A.F. (Eds.), Invertebrate Blood Cells I. Academic Press, London, pp. 233–299. Cheng, W., Hsiao, I.S., Hsu, C.H., Chen, J.C., 2004. Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Fish Shellfish Immunol. 17, 235–243. Chu, F.E., La Peyre, J.F., 1993. Development of disease caused by the parasite, Perkinsus marinus and defense-related hemolymph factors in three populations of oysters from Chesapeake Bay, USA. J. Shellfish Res. 12, 21–27. Delaporte, M., Soudant, P., Moal, J., Lambert, C., Quéré, C., Miner, P., Choquet, G., Paillard, C., Samain, J.F., 2003. Effect of a monospecific algal diet on immune functions in two bivalves species Crassostrea gigas and Ruditapes philippinarum. J. Exp. Biol. 206, 3053–3064.
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