Starvation-induced changes of hemocyte parameters in the Zhikong scallop Chlamys farreri.
ABSTRACT Substantial nutritional and energetic demands are associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained in lantern nets in different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast, no significant effect of starvation was observed on reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control, whereas ACP activity in hemocyte lysate was significantly lower in the starved group. These results indicate that starvation stress compromises immunological activities of scallops.
KEY WORDS: immunology, scallop, aquaculture, Chlamys farreri
The hemocyte, considered to be the main cell mediator in molluscan internal defenses, is involved in nutrient digestion and transportation (Feng et al. 1977, Cheng 1981), and is influenced by the nutritional condition of the animals. Partial or complete food deprivation alters the biochemical composition of oysters, Crassostrea gigas (Thunberg) (Whyte et al. 1990), decreases their metabolic activity (Rodhouse & Gaffney 1984) and changes the hemocyte defense capacity (Oubella et al. 1993, Funakoshi 2000, Hegaret et al. 2004, Butt et al. 2007).
A relationship between nutritional input (quantity of food) and the ability of bivalves to maintain defense capabilities seems likely, with starvation stress as an extreme case. Thus far, few studies have addressed this topic (Delaporte et al. 2003, Hegaret et al. 2004, Butt et al. 2007). Delaporte et al. (2003) suggested that the fatty acid composition of monospecific algal diets affected membrane functions of the oyster, C. gigas, and the clam, Ruditapes philippinarum (Adams & Reeve, 1850), modification of the membrane lipid composition influenced the hemocyte functionality. Hegaret et al. (2004) demonstrated that starvation combined with high-temperature shocks changed the hemocyte defense capability of the oyster, Crassostrea virginica (Gmelin), resulting in an increased respiratory burst, and decreased phagocytosis, aggregation, and hemocyte count, thereby possibly making them more susceptible to disease and parasites. In the Sydney rock oyster, Saccostrea glomerata, parameters related to immunological defense, including hemocyte counts, phenoloxidase, superoxide, and peroxidase activities decreased significantly after several weeks of starvation (Butt et al. 2007). Thus, health indicators plus knowledge of the defense systems are considered to be keys to understanding and eventually avoiding health problems of molluscs in culture (Bachere et al. 1995).
The Zhikong scallop, Chlamys farreri, is one of the most important cultured molluscs in northern China. In 1996, Chinese aquaculture produced one million metric tons of scallops, about 75% to 80% of which were the Zhikong scallop (Guo et al. 1999). To further augment production, farmers have been growing scallops at increasing densities within lantern nets and adding more longlines to the culture areas; this could result in insufficient nutrition or even starvation in areas low in nutrients for scallop survival. Studies on the effects of starvation on survival rate, condition index, changes in the composition of different tissues, respiration, and excretion of C. farreri have been conducted by our team (Yang et al. 1999), the results showed that starvation has strong impact on the condition index, the oxygen consumption rate (OCR), and the ammonia-N excretion rate (AER) of scallops. However, no study has yet addressed the effects of food deprivation on the hemocyte parameters of C. farreri.
The aim of our present work is to assess the potential susceptibility of scallops to invasive pathogens under extreme starvation conditions, which can be used in future monitoring the susceptibility of scallops in culture to disease. We chose several immunological parameters of hemocytes in C. farreri as our immunomarkers, including the total hemocyte counts (THC), percentage of phagocytic hemocytes, reactive oxygen species (ROS) production, and acid phosphatase (ACP) and superoxide dismutase activities in cell-free hemolymph, and in hemocyte lysate.
MATERIALS AND METHODS
Scallops, Chlamysfarreri (4.5 [+ or -] 0.3 cm in shell height), were collected from Jiaozhou Bay (in April, 2006) and acclimated in ambient temperature, grit-filtered seawater for two weeks prior to treatment. During the acclimation period, scallops were maintained in lantern nets suspended in 800-L tanks containing aerated filtered seawater (30 [+ or -] 1 [per thousand] salinity) that was renewed daily, and were fed daily on microalgae Phaeodactylum tricornutum Bohlin at 0.12 x [10.sup.9] cells [scallop.sup.-1] [day.sup.-1], which previously was shown to exceed satiation (unpublished results).
After the acclimation period, two nutritional treatments (satiation and starvation) were established. One group of scallops was fed at the satiation level, and the other group was starved for 40 days. Each treatment used three replicate tanks. Scallops were arbitrarily distributed among 0.7-m long lantern nets with 5 layers suspended in each 800-L tank. Each layer was a growout compartment that was 30-cm in diameter and 12-cm in height. The nets were hung on a plastic pole with the top compartment 3-4 cm below the water surface. During the 40-day experiment, 50% of the seawater was renewed twice weekly; water was maintained between 15[degrees]C and 17[degrees]C and at 30 [+ or -] 1 [per thousand] salinity, with continuous aeration to supply oxygen. Microalgae Phaeodactylum tricornutum at 0.12 x [10.sup.9] cells [scallop.sup.-1] [day.sup.-1] were added daily to the satiation treatment tanks.
At the beginning of the experiment, 30 samples were arbitrarily collected from the acclimation tanks for condition index measurement and 15 pools of 30 scallops for each hemocyte parameter were analyzed, which were used as initial control values. At the end of the experiment (40 days), the scallops were arbitrarily sampled again following the protocol of the beginning.
Scallop condition index was assessed using the dry weight method (Yang et al. 1999). Scallop flesh was dried at 65[degrees]C until a constant weight was reached. The dry flesh weight was divided by the dry shell cavity volume and multiplied by 100 to provide a final condition index for each scallop.
Hemolymph 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.
Measurements of Hemocyte Parameters by Flow Cytometer
Immune parameters of C. farreri were measured using a FACSVantage (BD Biosciences) flow cytometer. As recommended by the manufacturer, samples were filtered through 50-[micro]m mesh to eliminate potential large debris that might clump in the flow cytometer. Methods to measure hemocyte parameters are described hereafter.
An aliquot of hemolymph was mixed 1:1 with anticoagulant solution (Glucose 20.8 g [L.sup.-1], EDTA 20 mM, Sodium chloride 20 g [L.sup.-1], Tris-HCl 0.05 M, pH = 7.4) to prevent hemocyte clotting and for use as hemolymph working solution for flow cytometer detection (Richard et al. 1997).
Hemocyte Functional Parameters Detection
The total hemocyte counts (THC), the percentage of phagocytic hemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities were detected according to the methods of Chen et al. (2007a, 2007b).
One-way Analysis of Variance (ANOVAs) were performed for condition index and all the immune parameters analyses with SPSS 11.5 statistical software. Multiple comparison (Tukey) tests were conducted to compare significant differences among treatments.
After 40 days treatment, the condition index of the starved group was significantly lower (Fig. 1, P < 0.05) than in the satiated group and initial control.
After 40 days treatment, THC was significantly lower in the starved group (average 1.97 x [10.sup.7] cells [mL.sup.-1]) compared with the satiated group (average 3.34 x [10.sup.7] cells [mL.sup.-1]) and the initial control (3.29 x [10.sup.7] cells [mL.sup.-1]; Fig. 2, P < 0.05). The percentage of phagocytic hemocytes was significantly lower in the starved scallops (average 11.99%) compared with those of satiated and initial control scallops (averages of 14.99% and 13.45%; Fig. 3, P < 0.05). Moreover, the percentage of phagocytic hemocytes was even significantly higher in the satiated group than in the initial control group (Fig. 3, P < 0.05).
No significant effects of food deprivation were observed on ROS production (Fig. 4, P > 0.05), ACP activity in cell-free hemolymph (Fig. 5A, P > 0.05), or SOD activities in hemocyte lysate and in cell-free hemolymph (Fig. 6, P > 0.05); however, ACP activity in cell-free hemolymph was significantly higher after 40 days treatment in satiated and starved scallops as compared with the initial control scallops (Fig. 5A, P < 0.05), whereas ACP activity in hemocyte lysate was significantly lower in the starved group than in the satiated and initial control groups (Fig. 5B, P < 0.05).
A trade-off between traits associated with immune function may be expected because substantial nutritional and energetic demands are associated with immune activation and the maintenance of an efficient immune system (Lochmiller & Deerenberg 2000). Invertebrates are believed to divert energy away from nonessential processes such as growth, reproduction, and certain immune functions and into particular bio-energetic processes, such as increased oxygen uptake and mobilization of energy substrates, which help the animals to adapt and overcome the stress (Lacoste et al. 2002). In the present study, we investigated the effects of starvation on condition index and immune parameters of C. farreri. Starvation and satiation treatment were conducted for 40 days in combination with the practice of aquaculture handling.
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Results presented in this study demonstrated that the condition index decreased significantly after 40 days of starvation, which is consistent with our previous research work (Yang et al. 1999). Many other studies also suggested the decline of condition index of the filter-feeding bivalves under nutrient stress (Gabbott & Bayne 1973, Robinson et al. 1981, Widdows 1978, Zhang et al. 1991). It is probably because of their filter-feeding habit, when less food in the nutrient lean seawater can be found, they may increase their filtering rate and clearance rate for surviving. As a consequence, more energy would be consumed, which supposed to be reserved in different tissues when suffered from nutrient stress (Yang et al. 1999).
Decreases in THC after starvation indicate that hemocytes are also influenced by the nutritional condition of the scallops. In bivalves, hemocytes are found in the vascular system and also wandering through tissues (a characteristic of "open" circulatory systems). Thus, they may absorb nutrients from the digestive gland and pass them directly to other tissues (Cheng et al. 1996). If the organism is subjected to long-term starvation, as in our study, this function may be reversed, which is in agreement with Oubella et al. (1993), who hypothesized a reversible mobilization of hemocytes from hemolymph into soft tissues to compensate for the lack of nutrients.
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Among hemocyte functions, phagocytosis is considered to be one of the main cellular functions of bivalve defense systems (Pipe et al. 1995). In our study, the percentage of phagocytic hemocytes decreased significantly in starved scallops compared with satiated ones, which may result from the low energy of hemocytes after long-term food deprivation, as proposed by Hegaret et al. (2004). This is supported by Delaporte et al. (2003), who suggested that hemocytes that have accumulated certain fatty acids, which are unavailable during starvation, could be more active during phagocytosis and metabolic processes. In addition, our previous research (Yang et al. 1999) showed that O:N ratios were lowest after 20 days starvation, which indicated high consumption of proteins during this period. In vertebrates, reduced leptin (a 16 kDa protein) levels after starvation was proposed to cause impaired immune capabilities (Lord et al. 1998, Faggioni et al. 2001). The same protein is also reported in molluscs (de Jong-Brink et al. 2001), although its functional mechanism needs further study.
ROS production is another important mechanism in bivalve cellular defense systems (Pipe 1992). In our study, 40 days starvation did not significantly affect ROS production, suggesting that the stress duration was insufficient to induce any modification of ROS production. Although Hegaret et al. (2004) reported starved oysters had higher ROS than satiated oysters, their results were not statistically significant. The weak relationship between starvation stress and ROS production indicates that different immunomarkers have different sensitivity to starvation stress, which may depend on several factors such as stress duration or species specificity.
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Starvation changes the homeostasis of animals, and also may affect lysosomal hydrolysis (Kolataj et al. 2004). In the present study, the significant increase of ACP activity in cell-free hemolymph from satiated and starved scallops after 40 days treatment, which correspond to the decrease of ACP activity in hemocyte lysate, may result from destabilization of lysosomal membranes induced by gametogenesis or food deprivation (Zhang & Li, 2006). In northern China, C. farreri is usually sexually mature from the middle of the May to July, when temperatures are high. On treatment day 40, the satiated scallops appeared to have stopped feeding and to have well-developed gonads, indicating imminent spawning.
SOD is well known to be the first and most important defense against damage from excessive ROS production (Downs et al. 2001). In the current study, SOD tended to decrease after 40 days starvation, but this difference was not statistically significant, which is consistent with the results for ROS production. All of these results suggest that it is necessary to extend the stress duration, which probably would show greater influence on scallop health conditions.
In conclusion, our present experiment demonstrated that the hemocyte parameters of scallops were influenced by food deprivation. THC, percentage of phagocytic hemocytes, and ACP activity responded clearly to starvation stress and may be useful as prospective and efficient immunomarkers to monitor starvation stress in scallops. The weak changes in ROS production and SOD activity probably resulted from perturbation of the reproductive cycle or insufficient stress duration. Therefore, additional research outside the reproductive period and extending the stress period will clarify these issues.
This research was supported by National Natural Science Foundation of China (No. 30671614), National Key Foundational Research Project of China (No. 2007CB407305), and Hi-tech Research and Development Program of China (No. 2006AA 100304/2006AA 100307).
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BIAO XU, (1,2) MUYAN CHEN, (1,2) HONGSHENG YANG (1) * AND SANJUN ZHAO (1,2)
(1) Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong Province, 266071, China; (2) Graduate University, Chinese Academy of Sciences, Beijing, 100049, China
* Corresponding author. E-mail: email@example.com