Seasonal variations of water temperature, food availability, size, and reproduction on the hemocyte parameters in the scallop Chlamys farreri.
Article Type: Report
Subject: Adaptation (Physiology) (Research)
Scallops (Physiological aspects)
Authors: Lin, TingTing
Zhou, Kai
Lai, Qifang
Yao, Zongli
Li, Ziniu
Xing, Jing
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 0913070 Scallops NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: China Geographic Code: 9CHIN China
Accession Number: 303011388
Full Text: ABSTRACT It is well known that immune defenses of bivalves against environmental and pathogenic stresses are primarily attributable to hemocytes. Hemocyte parameters are being used progressively as indicators to assess the host immune status. Moreover, there is increasing evidence that seasonal variations on the immune status have two origins: exogenous and endogenous. In this work, we investigated the effects of seasonal exogenous (water temperature and food availability) and endogenous (size and reproduction) factors on the hemocyte parameters in the scallop Chlamys farreri. Scallops were monthly collected from February to December 2009 at 2 sampling sites differing in culture mode: Qingdao for monoculture and Weihai for scallop-kelp polyculture. Six hemocyte parameters including total hemocyte count (THC), granulocyte percentage (GP), intrahemocytic phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD), and peroxidase (POD) were analyzed. Results illustrated that all hemocyte parameters exhibited marked seasonal variations, following a similar pattern at both sites. High values of THC, GP, PO, and POD were observed in spring and early summer, a period of favorable water temperature and high food availability and gonad index, whereas low values were found in summer and early autumn, a period corresponding to reproduction completion and high water temperature. Moreover, SOD was lowest in February and highest in August, and correlated positively with water temperature. Hemocyte parameters in the scallop C. farreri varied greatly among seasons, and their values were generally low during summer and early autumn, suggesting that scallops had a depressed immune status during this period.

KEY WORDS: scallop Chlamys farreri, hemocyte parameters, water temperature, food availability, reproduction, size

INTRODUCTION

The immune system of bivalves has been studied widely during the past 3 decades (Duchemin et al. 2007). It is an innate immune system that depends largely on the circulating cells, collectively known as hemocytes (Auffret 1988, Cheng 1996). Hemocytes are involved in primary immune responses, including aggregation (Hegaret et al. 2003, Aladaileh et al. 2007), nodule formation (Hine 1999), phagocytosis (Bayne 1990, Cheng 1996), wound healing (Franchini & Ottaviani 2000), and production of reactive oxygen species (ROS) and active enzymes that are well known to assist, opsonize, and accelerate hemocyte immune functions (Xing et al. 2008). Hemocytes have been classified into 2 types: granulocytes and hyalinocytes (Hine 1999). In general, granulocytes are the main immunoreactive cells whereas hyalinocytes are less phagocytic or nonphagocytic and produce fewer ROS than granulocytes (Auffret 1988, Hine 1999, Hegaret et al. 2003, Aladaileh et al. 2007). The status of the bivalve immune system can be estimated by assessing a number of hemocyte parameters (number of hemocytes, hemocyte mortality, phagocytic activity, intrahemocytic enzyme activity, and so forth) that indicates whether the components of the immune system are structurally capable of performing immune functions efficiently (Auffret 2005). Being both osmo- and thermoconformers, bivalve hemocyte parameters are affected easily by exogenous environmental factors such as water temperature, food availability, salinity, and pathogenic microbes (Fisher 1988, Paillard et al. 2004, Soudant et al. 2004, Gagnaire et al. 2006). In addition, endogenous factors such as reproduction, size, age, and growth have their effects as well (Carballal et al. 1998, Duchemin et al. 2007).

The scallop Chlamys farreri is usually distributed throughout the coast of the Shandong and Liaodong peninsulas, particularly in Qingdao, Yantai, Weihai, Rizhao, and Dalian, China. It is a eurythermal species, with the tolerant and optimum temperatures of -l-30[degrees]C and 15-17[degrees]C, respectively. It filter feeds mostly on phytoplankton (especially diatoms), zooplankton, bacteria, and detritus, and its reproductive period is during late May and early June (Guo et al. 1999). Culture of C. farreri was first developed in 1973 and has expanded greatly since then 1980s because of improved breeding technology, and has resulted in great economic benefits (Xiao et al. 2005, Guo & Luo 2006). However, as farm production has increased, there has also been an increase in scallop health problems. Large-scale mortality of C. farreri has persisted in recent summers namely, the period of reproduction completion--posing a serious threat to the scallop aquaculture (Fu et al. 2005, Xiao et al. 2005). Many studies have been carried out to investigate the causative etiology for this abnormal mortality, finding it is mainly caused by a combination of high water temperature, microbial pathogens, and energy depletion of reproduction (Xiao et al. 2005, Chen et al. 2007b, Xing et al. 2008, Lin et al. 2011).

With the goal of providing constructive data for the prevention or alleviation of C. farreri mortality, a few studies, such as improving host immunity (Lin et al. 2011), screening stress-related genes (Ma et al. 2009), maintaining reasonable stocking density (Zhang et al. 2006), and optimizing culture mode (Nunes et al. 2003, Yu et al. 2010), have begun to be carried out. However, the work on natural seasonal variations of the physiological status and potential defense capability in C. farreri is still lacking. In this study, we focused on the effects of seasonal factors (water temperature, food availability, size, and reproduction) on the hemocyte parameters [total hemocyte count (THC), granulocyte percentage (GP), intrahemocytic enzymes including phenoloxidase (PO) (EC 1. 14. 18.1), acid phosphatase (ACP) (EC 3. 1.3. 2), superoxide dismutase (SOD) (EC 1. 15. 1. 1), and peroxidase (POD) (EC 1.11.1.7)] in C. farreri, with the aim of collecting data for understanding more completely the difference in immune status among seasons, and the interactions between the environment and the host.

MATERIALS AND METHODS

Animals and Sampling Sites

The scallop Chlamys farreri was cultured in 2 unpolluted sites of Qingdao (36[degrees]03' N, 120[degrees]18' E)and Weihai (37[degrees]10' N, 122[degrees]25' E; Fig. 1). These 2 sites were different in culture mode: Qingdao for monoculture and Weihai for scallop-kelp (Laminaria japonica Aresch) polyculture. The scallops at both sites were placed in 2-m-long lantern nets with 10 layers. Each layer was a grow-out compartment that was 35 cm in diameter and 20 cm in height. The nets were hung on suspended longlines supported by rubber buoys so that the top compartment was 5 m below the surface. From February to December 2009, 5 lantern nets at each site and 15 scallops per layer from each lantern net were sampled monthly. Scallops within the same lantern net were mixed (n = 150) as 1 unit or 1 replication. After sampling, scallops were transported in fiberglass tanks containing aerated seawater from the sampling sites. Transportation time between field collection and laboratory processing was short: for Qingdao, 1.5 h; for Weihai, 3 h. Sampled scallops were processed immediately on arrival to the laboratory.

Seasonal Factor Monitoring

Water Column Parameters

Salinity, pH, water temperature, and chlorophyll a (indicator of food availability) were measured using a Yellow Springs Instruments 6600 V2 multiparameter sonde at each site and each month during sampling.

[FIGURE 1 OMITTED]

Shell Height and Gonad Index

Forty individual scallops selected randomly from 1 unit (n = 150) were used for shell height and gonad index measurement. The shells and soft tissues of each individual scallop were separated carefully, and the shell height (indicator of size) was measured using a vernier caliper. After that, gonads were separated from soft tissues and were oven-dried together with the remaining soft tissues to a constant weight at 60[degrees]C to calculate the gonad index (GI; indicator of gonadal development).

GI = GDW/TDW X 100%

where GDW is the gonad dry weight and TDW is the total dry weight of soft tissues (Guo & Luo 2006).

Hemocyte Parameter Analysis

Total Hemocyte Count and Granulocyte Percentage

Thirty other individual scallops selected randomly from the remaining scallops (150 - 40 = 110) in 1 unit were used for THC and GP determination. THC and GP in hemocyte suspension were analyzed using a Countess automated cell counter (Invitrogen, Grand Island, NY) and flow cytometer (Becton Dickinson, Franklin Lakes, NJ), respectively. Hemocyte suspension was prepared as follows. One milliliter of hemolymph from each scallop was withdrawn from the adductor muscle sinus with a sterilized syringe and was mixed simultaneously (1:1) with precooled anticoagulant (modified Alsever's solution; 0.12 M glucose, 0.03 M sodium citrate, 9 mM EDTA, 0.38 M sodium chloride, pH 7.2). The mixture of hemolymph and anticoagulant was then centrifuged at 700g for 10 min at 4[degrees]C to collect the hemocyte pellet. The pellet was suspended in 1 mL 0.01 M phosphate buffered saline (0.14 M sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogen phosphate dodecahydrate, 1.5 mM potassium phosphate monobasic, pH 7.4).

Acid Phosphatase, Superoxide Dismutase, and Peroxidase

Forty other individual scallops selected randomly from the remaining scallops (150 - 40 - 30 = 80) in 1 unit were used for ACP, SOD, and POD detection. One milliliter of hemolymph from each scallop was withdrawn and prepared into a hemocyte suspension according to the previously mentioned procedures. The hemocyte suspension was sonicated further (VCX 750; Sonics & Materials, Newtown, CT) with a sonication intensity of 30% and 5-sec pulses for 4 min at 4[degrees]C, then was centrifuged at 5,500g for 10 min at 4[degrees]C. After that, the resulting supernatant was collected as hemocyte lysate supernatant (HLS) for ACP, SOD, and POD detection.

ACP activity was measured by using p-nitrophenyl phosphate as substrate (Gonzalez et al. 1994). Briefly, a 100-[micro]L aliquot of HLS was mixed with 2.0 mL 0.1 M sodium acetate buffer (pH 5.0) containing 5 mM p-nitrophenyl phosphate. The reaction mixture was incubated at 37[degrees]C for 30 min, then 2.0 mL 0.1 M NaOH was added to terminate the reaction, and absorbance was measured spectrophotometrically at 405 nm, using p-nitrophenol as the standard. One unit of enzyme activity was defined as the production of 1 mg p-nitrophenol in 30 min at 37[degrees]C per mg protein. SOD activity was determined using the xanthine oxidase method described by McCord and Fridovich (1969), with modifications. Briefly, 50 [micro]L HLS was incubated for 5 min at 25[degrees]C with 20 mM potassium phosphate, 1.0 mM EDTA, 0.25 mM xanthine, and 0.17 mM cytochrome c (pH 7.8). The reaction was initiated by adding xanthine oxidase (0.16 U), and was assayed by monitoring the reduction of cytochrome c at 550 nm for 5 min (25[degrees]C) in the presence or absence of xanthine oxidase and SOD. One unit of enzyme activity was defined as the amount of SOD that inhibited the rate of cytochrome c reduction by 50%, and was expressed as units per mg protein.

POD activity was measured by monitoring the time course of the change in absorbance at 478 nm by guaiacol oxidation (Chance & Maehly 1956). The final reaction mixture contained 750 [micro]L 20 mM guaiacol, 200 [micro]L HLS, and 1.6 mL 16 mM [H.sub.2][O.sub.2] in acetate buffer (pH 3.8). One unit of enzyme activity was defined as the conversion of 1 [micro]g substrate by per mg protein per minute under the optimal reaction conditions.

Phenoloxidase

Thirty other individual scallops selected randomly from the remaining scallops (150 - 40 - 30 - 40 = 40) in 1 unit were used for PO analysis. One milliliter of hemolymph from each scallop was withdrawn and mixed with anticoagulant. After centrifugation, the resulting hemocyte pellet was suspended in 1 mL cacodylate buffer (0.05 M sodium cacodylate, 5 mM calcium chloride, 0.15 M sodium chloride, pH 7.5). Hemocyte suspension was sonicated and then centrifuged to collect the resulting supernatant as PO HLS for PO analysis.

PO activity was measured by the L-3,4-dihydroxyphenylalanine (L-dopa) transformation to dopachrome method (Soderhall 1981). A 100-[micro]L aliquot of PO HLS was incubated for 10 min at 20[degrees]C with 0.1 mL SDS, after which 2.0 mL substrate (L-dopa in Tris-HCl buffer) was added. The dopachrome formed was measured spectrophotometrically at 490 nm every 3 min for 30 min. One unit of enzyme activity was defined as an increase in absorbance of 0.001 per mg protein per minute.

Hemocyte Lysate Supernatant Protein

HLS protein concentration was determined by the Bradford (1976) method, using the Coomassie brilliant blue G-250 assay reagent. A total of 50 [micro]L HLS was mixed with 50 [micro]L 0.15 M NaCl, and then 1 mL Coomassie brilliant blue G-250 assay reagent was added. After 5 min, absorbance was measured spectrophotometrically at 595 nm, and the unit was expressed as mg protein per milliliter. A standard curve was developed using bovine serum albumin.

Statistical analysis

All data were presented as mean [+ or -] SD, and were checked for normal distribution (Shapiro-Wilk's test) and homogeneity of variances (Bartlett's test). The effects of sampling sites and sampling months on hemocyte parameters were examined using a 2-way ANOVA analysis followed by a post hoc test, and the relationships between seasonal factors and hemocyte parameters were evaluated using a Pearson's correlation test. In addition, if ANOVA assumptions were not fulfilled, the nonparametric Mann-Whitney U-test was used for pairwise comparisons between the 2 sites complemented with permutational analysis of variance. The level of statistical significance was defined as P < 0.05.

RESULTS

Seasonal Factors

Water Column Parameters

Salinity and pH at both sites were consistent throughout the entire sampling period, ranging from 29.04 [+ or -] 0.02 to 30.26 [+ or -] 0.03 [per thousand] and from 7.9 [+ or -] 0.15 to 82 [+ or -] 0.21, respectively (data not shown). Water temperature and chlorophyll a varied greatly among seasons, following a similar pattern at both sites. Water temperature increased from a minimum (2.8 [+ or -] 0.7[degrees]C for Qingdao, 2.6 [+ or -] 0.6[degrees]C for Weihai) in February, and reached a maximum (26.2 [+ or -] 1.3[degrees]C for Qingdao, 25.8 [+ or -] 1.4[degrees]C for Weihai) in August, then decreased, reaching about 10.3 [+ or -] 0.9[degrees]C in December (Fig. 2A). Chlorophyll a also increased from February, and reached a maximum (4.38 [+ or -] 0.33 [micro]g/L for Qingdao, 7.44 [+ or -] 0.41 [micro]g/L for Weihai) in June, and then decreased, reaching a minimum (1.08 [+ or -] 0.23 [micro]g/L for Qingdao, 1.15 [+ or -] 0.41 [micro]g/L for Weihai) at the end of the sampling period (Fig. 2B). Chlorophyll a levels in Weihai were obviously greater than those in Qingdao, particularly from April to September.

Shell Height and Gonad Index

Shell height increased continuously from February (2.5 [+ or -] 0.25 cm for Qingdao, 2.35 [+ or -] 0.24 cm for Weihai) to December (7.0 [+ or -] 0.38 cm for Qingdao, 7.12 [+ or -] 0.34 cm for Weihai), and its increase rates (indicator of growth) in April, May, June, and July were higher than those in other months. In addition, shell height in Weihai was generally higher than that in Qingdao from May to December, a period corresponding to higher chlorophyll a in Weihai than in Qingdao (Fig. 2C).

Gonad index also varied greatly among seasons, following a similar pattern at both sites. In March, scallop gonads were small, with a length of about 1.2 cm, with a gonad index of 9.24 [+ or -] 1.51% for Qingdao and 9.45 [+ or -] 1.25% for Weihai (Fig. 2D). The sexes were easily distinguished according to their color, with females being bright orange and males being ivory. From April to June, scallop gonads showed a continuous increase in length and gonad index with increasing water temperature and chlorophyll a. In June, scallop gonads were well developed, with a length of about 2.8 cm, with a maximum gonad index of 21.5 [+ or -] 1.60% for Qingdao and 22.8 [+ or -] 1.25% for Weihai (Fig. 2D). After June, the length and gonad index declined sharply as a result of spawning or spermiation (Fig. 2D), and by August, scallop gonads were shriveled and transparent, which resulted in difficulty distinguishing their sex. The minimum gonad index was 4.26 [+ or -] 1.21 % for Qingdao and 4.09 [+ or -] 1.20 for Weihai (Fig. 2D). Thereafter, scallop gonads began to recover; at the end of the sampling period, they were semitransparent, with a length of about 2.0 cm and a gonad index of 7.89 [+ or -] 1.25% for Qingdao and 7.47 [+ or -] 1.31% for Weihai (Fig. 2D). Their sex could be distinguished again, with females being slight orange, males being slight ivory.

[FIGURE 2 OMITTED]

Hemocyte Parameters

Total Hemocyte Count and Granulocyte Percentage

THC varied greatly among seasons in both sites, following a pattern similar to gonad index (Fig. 3A, P < 0.05). The values were intermediate in February and March, and increased rapidly thereafter, reaching the highest levels (32.5 [+ or -] 1.45 x [10.sup.6] cells/mL for Qingdao and 34.9 [+ or -] 1.55 x [10.sup.6] cells/mL for Weihai) in June, followed by a remarkable decrease in July and August. From August to November, the values remained constant, with the lowest levels of the year. In December, the values rebounded and were close to the initial levels (P> 0.05). For GP at both sites, the values in April, May, and June were significantly higher than those in the remaining months, which basically maintained stable, with mean levels of about 57% (Fig. 3B, P < 0.05). In addition, the GP value in Weihai was significantly higher than that in Qingdao during June (P < 0.05).

Phenoloxidase

PO activity changed significantly with seasons, following a similar pattern at both sites (Fig. 3C, P < 0.05). The values were intermediate at the beginning of the year, but increased dramatically thereafter and reached the highest levels (3.81 [+ or -] 0.22 U/mg protein/min for Qingdao, 3.57 [+ or -] 0.24 U/mg protein/ min for Weihai) in April, followed by a sharp decrease in May, June, and July. PO activity was lowest (0.98 [+ or -] 0.19 U/mg protein/min in August for Qingdao, 1.04 [+ or -] 0.20 U/mg protein/min in September for Weihai) from July to November. In December, the value rebounded to the level seen at the beginning of the year (P > 0.05).

Acid phosphatase

ACP activity varied greatly among seasons, following a similar pattern at both sites (Fig. 3D, P < 0.05). Except for a significant peak in May (73.76 [+ or -] 2.61 mU/mg protein for Qingdao, 82.25 [+ or -] 2.81 mU/mg protein for Weihai) and June (68.7 [+ or -] 2.74 mU/mg protein for Qingdao, 73.81 [+ or -] 2.64 mU/mg protein for Weihai), ACP activity showed a continuous increase with increasing size. In addition, the value of ACP activity in Weihai during May, June, and November was significantly higher than that in Qingdao (P < 0.05).

Superoxide dismutase

SOD activity changed significantly with seasons, following a similar pattern at both sites (Fig. 3E, P < 0.05). The lowest values were found in February (31.25 [+ or -] 3.11 U/mg protein for Qingdao, 30.45 [+ or -] 3.28 U/mg protein for Weihai) and March (33.21 [+ or -] 3.03 U/mg protein for Qingdao, 34.57 [+ or -] 2.89 U/mg protein for Weihai), followed by a progressive increase with increasing water temperature, and reached the highest levels (80.44 [+ or -] 3.89 U/mg protein in August for Qingdao, 80.36 [+ or -] 4.12 U/mg protein in September for Weihai) in summer and early autumn, and finally fell with decreasing water temperature in November and December. The association between water temperature and SOD activity was demonstrated by Pearson's correlation test. The values of this correlation coefficient were [R.sub.s] = 0.873 for Qingdao and [R.sub.s] = 0.911 for Weihai (n = 11, P < 0.05).

[FIGURE 3 OMITTED]

Peroxidase

Similarly, POD activity varied greatly among seasons in both sites, following a similar pattern to PO activity (Fig. 3F, P < 0.05). The values were moderate at the beginning of the year, but increased rapidly thereafter, reaching the highest level (58.60 [+ or -] 2.17 U/mg protein/min for Qingdao, 55.67 [+ or -] 2.43 U/mg protein/min for Weihai) in April, followed by a rapid decrease in May and June. From July to December, the values remained constant with the lowest levels of the year.

DISCUSSION

Bivalve hemocytes are responsible for primary immune responses, such as phagocytosis, encapsulation, oxidative killing, wound healing, and even for shell mineralization (Bayne 1990, Franchini & Ottaviani 2000, Hegaret et al. 2003, Mount et al. 2004). Endogenous enzymes, which are very important participants in the immune system, are well known to assist, modulate, and accelerate immunological processes in hemocytes. For example, SOD and POD, 2 typical antioxidases, are able to eliminate excessive ROS to alleviate the damage to hosts (Xing et al. 2008). ACP is capable of assisting, modulating, and accelerating phagocytosis, and is also involved in nutrient transport and digestion (Chen et al. 2007b). PO is an indispensable component of melanotic encapsulation (Soderhall & Cerenius 1998). Bivalves are frequently exposed to environmental factors such as water temperature, salinity, dissolved oxygen, nutrients, toxicants, and parasites that exhibit intermittent and short-/long-term fluctuations, and their cellular defense-related parameters are known to be especially sensitive to the disturbance of these factors (Ballarin et al. 2003, Auffret et al. 2004, Matozzo et al. 2008). In the current study, we also validated that the hemocyte parameters in the scallop C. farreri varied greatly among seasons. High values of THC, GP, PO, and POD were observed in spring and early summer, a period of favorable water temperature by C. farreri, and high food availability and gonad index, whereas low values were found in summer and early autumn, a period corresponding to reproduction completion and high water temperature.

Low values of THC and GP are considered widely to be a sign of disturbance in the oysters Crassostrea virginica (La Peyre et al. 1995) and Saccostrea glomerata (Butt et al. 2007), the mussel Mytilus edulis (Pipe & Coles 1995, Parry & Pipe 2004), the clam Mactra veneriformis (Yu et al. 2009), and C. farreri (Chen et al. 2007a, Xu et al. 2008). On the other hand, the activity of intrahemocytic enzymes of bivalves is used increasingly to assess the host's physiological and immunological status (Auffret et al. 2006). Taken as a whole, high levels of intrahemocytic enzyme activity indicate a good immune capability (Galloway & Depledge 2001, Duchemin et al. 2007, Lin et al. 2011), and a decrease of intrahemocytic enzyme activity is usually observed in laboratory studies as a response of hemocyte disturbances resulting from in vivo or in vitro exposures to environmental stresses and pathogenic microbes (Butt et al. 2007, Chen et al. 2007b, Xu et al. 2008, Morga et al. 2009). Consequently, the current results give evidence of a prosperous immune status during spring and early summer, and a depressed one during summer and early autumn in C. farreri.

It is well documented that numerous seasonal factors have an impact on the hemocyte parameters of bivalves (Santarem et al. 1994, Duchemin et al. 2007), among them, water temperature and food availability are recognized as two of the most decisive exogenous factors that are responsible for bivalve metabolism, growth, gonad development, hemocyte motility, and so forth (Fisher 1988, Carballal et al. 1998, Delaporte et al. 2006, Monari et al. 2007, Strohmeier et al. 2008). There is increasing evidence that the reproductive cycle also has an effect on the hemocyte parameters of bivalves (Carballal et al. 1998, Duchemin et al. 2007, Li et al. 2009). Hemocyte migration toward gonadal interstitial tissue, and finally, toward gonadal follicles has been observed extensively during gametogenesis (Cajaraville et al. 1996). In this study, high values of THC, GP, PO, and POD were observed during the period of favorable water temperature by C. farreri, and high food availability and gonad index, whereas low values were found during the period corresponding to reproduction completion and high water temperature. Furthermore, SOD correlated positively with water temperature. These results give evidence of the decisive effects on hemocyte parameters by water temperature, food availability, and reproduction. Similar results were observed in other bivalves. In oysters C. virginica and Crassostrea gigas, high hemolymph protein and lysozyme levels were found during the period of high food availability (Chu & La Peyre 1989), and high hemocyte mortality and low phagocytic index were detected during the period of postspawning and high water temperature (Duchemin et al. 2007). However, in the mussel Mytilus galloprovincialis, low GP and THC were observed, respectively, during a period of parasitism and high water temperature (Santaram et al. 1994). In this study, we also found that, except for a significant peak in May and June, a period corresponding to high food availability, ACP activity showed a continuous increase with increasing size, which may suggest that in addition to immune defense, ACP in C. farreri is involved in functions such as food ingestion, nutrient transport and digestion, and even shell mineralization (Carballal et al. 1998, Jing et al. 2006).

High water temperature can depress hemocyte parameters in bivalves, including hemocyte number, motility, viability, adhesive capacity, phagocytic ability, membrane permeability, and intracellular enzyme activity, which results in a weakened ability to mount an immune defense (Fisher 1988, Le Moullac & Haffner 2000, Monari et al. 2007, Linet al. 2011). Spawning or spermiation, a physiological process of high energy cost, has also been reported to exhibit an immunosuppressive effect on hemocyte parameters (Duchemin et al. 2007). Many studies illustrate that spawned C. gigas are generally in a fragile condition, with low levels of glycogen and tissue proteins (indicators of energy reserve); low hemocyte concentrations, phagocytic activity, and adhesive capacity; and high hemocyte mortality (Delaporte et al. 2006, Duchemin et al. 2007, Li et al. 2009). Therefore, in this article, the depressed immune status of C. farreri observed during summer and early autumn can be attributable primarily to the interaction of high water temperature and high energy cost by reproduction. But, because the disease rate of C. farreri is elevated during this period (Xiao et al. 2005, Xing et al. 2008), we cannot exclude that this depressed immune status may be related to pathogenic interference as well. In addition, some studies found that bivalves with a weak health condition during summer and early autumn was also the result of nutritional stress (Soudant et al. 2004, Mitra et al. 2008). Given that food availability was still high during this period, the depressed immune status of C. farreri and whether it is caused by nutritional stress should be studied further by determining the condition index, glycogen and lipid content, and so on.

In the current study, we also observed that chlorophyll a concentration was nearly two times higher in Weihai than in Qingdao during the spring and summer. This difference was mainly because Weihai is closer than Qingdao to the mouth of the Yellow River and Bohai Bay, where a lot of phytoplankton accumulates each spring and summer. However, the difference in food availability, together with the different culture mode, did not result in a significantly different pattern in hemocyte parameters between these two studied sites. The explanation may be associated with the phytoplankton species, according to the data provided by Zhang (2010). The phytoplankton species compositions in Qingdao and Weihai were quite similar, being composed of 50% Bacillariophyta, 29% Pyrrophyta, 5% Chlorophyta, 5% Chrysophyta, 7% Protozoa, and 4% unidentified species.

In conclusion, hemocyte parameters of the scallop C. farreri at both sites exhibited marked seasonal variations. Values were generally low during the summer and early autumn, a period of reproduction completion and high water temperature, suggesting that scallops had a depressed immune status during this period as a result of the interaction of high water temperature and the high energy cost of reproduction. Although this study cannot be used to assess which seasonal factors have the greatest impact on scallop immune status, it provides valuable information on evaluating scallop growth performance and disease susceptibility among the culture seasons.

ACKNOWLEDGMENTS

We are grateful to Professor Chongming Wang (Yellow Sea Fishery Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China) for sample collection. This research was supported by the NSFC (grant no. 30901112), the National 863 Project (grant no. 2006AA100307), and the Agriculture Ministry Project (grant no. 200803012-04).

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TINGTING LIN, (1,2) KAI ZHOU, (1) QIFANG LAI, (1) ZONGLI YAO, (1) ZINIU LI (1,2) AND JING XING (2) *.

(1) East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation, Ministry of Agriculture, Shanghai 200090, PR China; (2) Laboratory of Pathology and Immunology of Aquatic Animals, LMMEC, Ocean University of China, Qingdao 266003, PR China

* Corresponding author. E-mail: xingjingouc@yahoo.com.cn

DOI: 10.2983/035.031.0309
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