Aquaculture Research, 2007, 38, 1380 ^ 1386 d o i: 10.1111/ j.1365 -2109.20 07.01813. x

Effects of stocking density on growth and non-specific immune responses in juvenile soft-shelled , Pelodiscus sinensis

Xinran Chen1,2, Cuijuan Niu1 &LijunPu1,3 1Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing, PR China 2Chinese Academy of Fishery Sciences, Beijing, PR China 3Life Sciences and Biotechnology Institute, Heilongjiang August First Reclamation University, Daqing, PR China

Correspondence: Cuijuan Niu, Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing 100875, PR China. E-mail: [email protected]

Abstract Introduction To investigate the in£uence of stocking density on the A high stocking density (StD) is one of the most com- growth and non-speci¢c immune responses of juvenile mon stressors in aquaculture, which elicits a wide soft-shelled turtle, Pelodiscus sinensis, three groups of variety of physiological responses in aquatic (initial body weight 74.2 12.2 g) were reared such as alterations in poor feed utilization (Stone, in 80 cm (L) 40 cm (W) 30 cm (H) tanks for 35 Allan & Anderson 2003), poor growth and mobiliza- days at three stocking densities (StD), which were 1 tion of energy sources in animals (Robert1998; Burns individuals (ind.)tank 1 (3.13 ind m 2)forD1,4 2000) or even mortality (Suomalainen, Tiirola & ind. tank 1 (12.5 ind m 2) for D2 and 6 ind. tank 1 Valtonen 2005). Crowding also in£uences immune (18.75 ind m 2) for D3 respectively.D1was the control function. It can reduce serum complement (Rotllant, group.Turtles were weighted individuallyat the begin- Pavlids & Kentouri1997; Montero, Marrero, Izquierdo, ning and at the end of the trial. At the end of the rear- Robaina, Vergara & Tort 1999) and depresses the ing trial, non-speci¢c immune parameters were phagocytic function (Yin, Lam & Sin 1995; Iguchi, determined. The results showed that the heterophil:- Ogawa, Nagae & Ito 2003), indicating immunosup- lymphocyte ratio (HLR) increased and the total plasma pressive e¡ects. But changes in lysozyme activity var- protein decreased with increasing StD, suggesting a ied after chronic crowding (Rotllant et al.1997; stress response. Speci¢c growth rate (SGR) signi¢- Montero et al.1999;Wang,Wang, Li & Cai 2004). In£u- cantly decreased with increasing StD. Non-speci¢c im- ence on the immune system is very important for mune indicators such as blood cell phagocytic activity, aquatic animals, because its impairment may lead to serum haemolytic activity and serum bacteriolytic disease outbreaks or death, especially under crowded activity increased clearly at a higher StD, indicating a rearing conditions. Hence, establishing an appropri- pattern of immunoenhancement.The results indicated ate StD for better growth and immune function is of that at StDs of 4 and 6 ind. tank 1, non-speci¢c im- great importance to successful aquaculture. mune responses were not suppressed compared with Soft-shelled turtle Pelodiscus sinensis is an impor- the control, but were inversely enhanced at the cost of tant aquaculture species in China. However, few growth reduction. A possible energy-based trade-o¡ reports have dealt withthe e¡ects ofcrowdingonthis between growth and constitutive immunity may exist species.The present studyaimed to investigate in soft-shelled turtles. the e¡ect of stocking density on the immune status of the turtle juveniles. Because the ’ antibody Keywords: stocking density, non-speci¢c immune responses to antigenic stimulus are much slower responses, growth, soft-shelled turtle, Pelodiscus than that of birds or mammals (Jurd 1994; Shen, sinensis Zhang, Quan, Liu & Yin 2003), their defences against

r 2007 The Authors 1380 Journal Compilation r 2007 Blackwell Publishing Ltd Aquaculture Research, 2007, 38, 1380^1386 Stocking density e¡ects on immunity of soft-shelled turtle X Chen et al. pathogen depend more on non-speci¢c immunity ately placed into heparinized tubes and non-hepari- (Alexander & Ingram 1992). Therefore, we focused nized tubes. Heparinized blood was for phagocytosis on non-speci¢c immune functions as monitoring in- analysis and sera were collected from non-hepari- dicators to evaluate the in£uence of crowding stress. nized blood for alternative complement pathway The stocking density setting in the study was set up (ACP) activity and bacteriolytic activity analysis. A in consultation with culturists based on their empiri- drop of blood from each turtle was smeared on a cal experiences. microscope slide, air-dried, ¢xed with absolute methanol and stained using a Wright^Giemsa Materials and methods stainer. Heterophils and lymphocytes were di¡eren- Experimental animals and treatment tiated out of 300 cells counted at a magni¢cation of 1000. The heterophil:lymphocyte ratios (HLR) In November 2004, juvenile soft-shelled turtles were were calculated for each turtle. Body weight was obtained from a turtle farm in Beijing and were ran- measured after a 48-h food deprivation at the begin- domly distributed into 64 L aquaria (three turtles per ning and at the end of the experiment. tank) with19L tap water inside. The photoperiod was a natural light^dark cycle of12L:12D.Turtles were fed to apparent satiation once daily at 15:00 hours. Unea- Analytical methods ten food was removed 1h later and one-fourth of the The phagocytic activity of whole blood cells was ana- water in each tank was replaced with equal-tempera- lysed using the yeast, Saccharomyces cerevisiae,asan ture dechlorinated water. The rearing water was indicator according to the method of Zhou, Niu, Sun maintained at a temperature of 29 1 1CpH7.6^7.8, and Li (2002). Dried live yeast was incubated in 2% su- and the total ammonia was less than10mg L 1. crose solution (pH 3^4) for 2 h at 30 1C and boiled for After acclimation for 3 weeks to the laboratory 30 min.Then, the suspending yeast was centrifugated environment, healthy animals (body weight and the pellet was washed twice and resuspended in 74.2 12.2 g) were selected (sex was not considered) 0.85% saline. Then, 20 mL2 10 8 cell mL 1 yeast and assigned randomly to three experimental den- suspension as well as 40 mL heparinized whole blood sity groups as follows: 1individuals (ind.)tank 1 were added to a 0.1mL Eppendo¡ tube. The mixture (3.13 ind m 2)forD1,4ind.tank1 (12.5 ind m 2) was incubated at 30 1C for 30 min with gentle shaking for D2 and 6 ind. tank 1 (18.75 ind m 2)forD3. at intervals, and then Wright^Geimsa-stained blood There were no signi¢cant di¡erences (ANOVA: smears were prepared. The number of blood cells that F 52.356, P40.05) in body weight among all 2, 51 were phagocytizing yeast was determined out of 300 treatment groups (Table 1). Other rearing conditions blood cells under 1000 power,and replicate smears were kept the same as those during the acclimation were observed for each turtle. The phagocytic activity period. During the experiment, we replaced a dead of blood cells represented by phagocytosis percentage turtle with another iso-weight healthy turtle to was calculated as: maintain a consistent StD. phagocytosisð%Þ¼the number of blood cells containing yeast=300 100%: Sampling The serum haemolytic activity induced by an At the end of the experiment, eight turtles from each ACP was determined according to the method of group were decapitated quickly. Blood was immedi- Sunyer and Tort (1995) with a slight modi¢cation.

Table 1 Speci¢c growth rates (mean SE) of Pelodiscus sinensis held at di¡erent stocking densities

Parameters

Stock density Initial body weight g 1 Final body weight g 1 SGR/% day 1w

D1Ã 70.09 3.39 94.71 17.21 0.89 0.14a D2Ã 73.96 2.26 87.73 18.10 0.51 0.08ab D3Ã 78.34 3.09 84.68 15.65 0.20 0.04b

ÃStocking densities: 1ind. tank 1 (3.13 ind m 2)forD1,4ind.tank1 (12.5 ind m 2)forD2and6ind.tank1 (18.75 ind m 2)forD3. wMeans denoted by di¡erent superscript letters in the same columns were signi¢cantly di¡erent (Po0.05).

r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386 1381 Stocking density e¡ects on immunity of soft-shelled turtle XChenet al. Aquaculture Research, 2007, 38, 1380^ 1386

Twenty-¢ve microlitres (1%) rabbit blood cells (RBC) plicates were measured per turtle. Bacteriolytic ac- suspension was added to100 mL serially diluted (from tivity (UI) was estimated by the following formula: 1:40 to 1:16) turtle serum in Mg 21EGTA-GVB [0.1% Bacteriolytic activity (UI) ¼ðA0 AÞ=A: gelatin, 0.1mol L 1 ethylene glycol tetraacetic acid 1 Total plasma protein was determined according to (EGTA) and 0.1mol L MgCl2] to make the ¢nal vo- lume of 125 mL. On the other hand, 25 mL 1% rabbit the Bradford method (Bradford1976). All assays were RBC suspension and 25 mL reciprocal-diluted sample performed in duplicate, and the mean SE were cal- serum were added, respectively, to 100 mLMg21EG- culated for each group (n 512). The removed spleen TA-GVB as controls. The tubes were incubated at was weighted with electronic scales (d 5 0.1mg). The 30 1C for 15min, and shaken several times during splenic index of individual turtles was calculated as the incubation. Finally,1mL EDTA-GVB (0.1% gelatin follows: and 0.1mol L 1 EGTA) was added to terminate the Spleen index ¼ spleen weight (g)=body weight (g) haemolytic reaction and then the mixture was cen- trifuged at 4 1C,664 g for 5 min.The extent of haemo- lysis was estimated by measuring the optical density Data analysis (OD) of the supernatant at 414 nm (OD414 nm). Abso- lute haemolysis (100%) was given by the optical read- Growth, indicated by the speci¢c growth rate (SGR) ing of the supernatant of a 25 mL RBC suspension to (%day 1), was calculated using the formula:

110 0 mL distilled water. The reciprocal of the serum SGR 5 (lnW2 lnW1)/(t2 t1) 10 0, wh e r e l nW2 dilution causing 50% haemolysis of RBC was desig- and lnW1 are the natural logarithms of the wet body nated the ACH50 titre, and the result was presented weights (g) at time t2 and t1 respectively. The di¡er- as ACH50 units per millilitre serum. The valueY (per- ences in SGR among groups were evaluated using centage of RBC lysed at each dilution) was de¢ned as univariate analysis with the initial weight as a cov- ariate. The least-signi¢cant di¡erence (LSD) test was Y ¼fAbs414ðAÞ½Abs414ðBÞ used to determine which speci¢c pairs di¡ered. The þ Abs414ðCÞg=½Abs414ðDÞAbs414ðBÞ relationship between the initial biomass StD and whereA is the supernatant of the test serum dilution, SGR was analysed using linear regression analysis. B is the control supernatant of the bu¡er containing The di¡erences in immune parameters among ex- the RBC without the serum, C is the control serum perimental groups were evaluated using one-way dilution without RBC and D is 100% haemolysis. Y analysis of variance (ANOVA), followed by the LSD mul- values were treated according to the Van Krogh tiple-range test when equal variance was assumed. transformation.Thus, the valuesY/(1 Y)andthere- When non-homogeneous variance was found, ciprocal of serum dilutions were plotted on a log^log Mann^Whitney and Kruskal^Wallis tests were used graph paper and the ACH50 was read from the graph. for the same purpose. The results were considered to Serum dilutions resulting in lysis greater than 90% be statistically signi¢cant at Po0.05. All data were or less than15% were excluded from the calculation. analysed using SPSS (version11.0) statistic software. The ability of the serum to lyse rabbit red blood cells (RRBC) was assayed because RRBC was a stimulus of the ACP. Results The serum bacteriolytic activity was measured using dried micrococcus, Micrococcus lysodeikticus, E¡ect of StD on growth as an indicator of serum lysis, following the methods The initial and ¢nal body weights and SGR are given described by Zhou et al.(2002).Micrococcus lysodeik- in Table 1. Stocking densities clearly showed a nega- ticus dried cells were suspended in ice cold tive in£uence on the growth of the turtle (ANOVA: 0.1mol L 1 potassium phosphate bu¡er pH 5 6.4 F2, 8 510.994, P 5 0.001o0.01; Table 1). Speci¢c (A 570 0.3). A 50 mL sample of serum was added to growth rate of the D3 group signi¢cantly decreased a 3 mL bacterial suspension in an ice bath, and the compared with the D1 and D2 groups (Po0.05). The absorbance (A) of this mixture was measured at negative linear relationship between StD and SGR 570 nm (A 0). The mixture was incubated at 30 1Cfor could be described as follows: 40 min, and then transferred back to the ice bath for SGR ¼0:275 StD þ 1:017 10min to stop the reaction. The absorbance of the 2 mixture was again measured at 570 nm (A). Two re- ðR ¼ 0:728; F1;9 ¼ 25:867; P <0:05Þ

r 2007 The Authors 1382 Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386 Aquaculture Research, 2007, 38, 1380^1386 Stocking density e¡ects on immunity of soft-shelled turtle X Chen et al.

Table 2 Immune parameters (mean SE) of Pelodiscus sinensis held at di¡erent stocking densities

Tr eat men t group

Immune parameter D1Ã D2Ã D3Ã

Blood cell phagocytic activity (%)w 1.094b 0.073 1.849a 0.280 1.135b 0.164 Serum hemolytic activit (ACH50 unit mL 1 serum)w 28.714b 5.089 30.676b 4.049 46.620a 5.125 Serum bacteriolytic activity (UI)w 0.290b 0.080 0.223b 0.036 0.867a 0.155

ÃStocking densities: 1ind. tank 1 (3.13 ind m 2)forD1,4ind.tank1 (12.5 ind m 2) for D2 and 6 ind. tank 1 (18.75 ind m 2) for D3. wMeans denoted by di¡erent superscript letters in the same lines were signi¢cantly di¡erent (Po0.05).

Table 3 Heterophil:lymphocyte ratios, total plasma protein and splenic indices (mean SE) of Pelodiscus sinensis at di¡erent stocking densities

Treatment group

Hematological parameter D1Ã D2Ã D3Ã

Heterophil:lymphocyte ratiosw 0.85 0.16c 1.73 0.27b 2.12 0.31a Total plasma protein (mg mL 1)w 28.10 0.45a 26.16 1.83ab 22.55 1.41b Splenic indices 0.0012 0.0012 0.0010 0.0001 0.0012 0.0001

ÃStocking densities: 1ind. tank 1 (3.13 ind m 2)forD1,4ind.tank1 (12.5 ind m 2) for D2 and 6 ind. tank 1 (18.75 ind m 2)forD3. wMeans identi¢ed by di¡erent superscript letters in the same lines were signi¢cantly di¡erent (Po0.05).

E¡ect of StD on non-speci¢c immune group having the lowest protein level (Po0.05, parameters Table 3, line 2). There were no signi¢cant di¡erences in the spleen indices among all the treatment groups Stocking density showed a signi¢cant in£uence on (ANOVA: F2, 21 51.046, P40.05;Table 3, line 3). blood cell phagocytosis of the turtle (ANOVA:

F2, 23 54.889, Po0.05).The blood phagocytic activity in the D2 group was signi¢cantly higher than the Discussion other two groups (Po0.05), while no signi¢cant dif- It has been reported that chronic stress could result ference was found between the D1 and D3 groups in a decrease in an ’s growth (Van Weerd & (P40.05) (Table 2, line1). The D3 group had signi¢- Komen, 1998; Foss, Siikavuopio, Saeher & Evensen cantly elevated serum haemolytic activity in relation 2004). Our result also showed a clear negative e¡ect to that of the D2 and D1groups (ANOVA: F 53.914, 2, 22 of StD on the growth of the turtle juveniles. Similar Po0.05; Table 2, line2), while there were no signi¢- results have been reported in many other species cant di¡erences between the D2 and D1 groups. The such as ¢sh (Canario, Condeca, Power & Ingleton serum bacteriolytic activity also had increased 1998), amphibian (Dash & Hota 1980) and avian clearly only in the highest StD D3 group (ANOVA: (Nephew & Romero 2003) after crowding. In this F2, 22 511.088, Po0.05;Table 2, line3) compared with study, stress responses were monitored by HLR, the D1and D2 groups. which have also been utilized as an indicator of gen- eral stress in chickens (Gross & Siegel 1983) and of disease in sea turtles (Aguirre, Balazs, Spraker & E¡ect of crowding on HLR, total plasma Gross 1995; Work, Rameyer, Balazs, Cray & Chang protein and splenic indices 2001). In the present result, an increase in the HLR The HLR? was signi¢cantly a¡ected by StD (ANOVA: indicated the severity of crowding stress in this ex-

F2, 17 56.470, Po0.05;Table 3, line1). Heterophil:lym- periment. phocyte ratio increased noticeably with StD and dif- Usually, two common units are adopted to denote fered signi¢cantly among the di¡erent StD groups. A turtle’s stocking density in the practice of aquicul- high StD signi¢cantly reduced the total plasma pro- ture, which are biomass m 2 and ind. m 2.Ina tein (ANOVA: F2, 21 55.353, Po0.05), with the D3 short period of time, the biomass changed slightly r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386 1383 Stocking density e¡ects on immunity of soft-shelled turtle XChenet al. Aquaculture Research, 2007, 38, 1380^ 1386 and the turtle number in the container acted as the explain the overall increase in the haemolytic activ- main factor in£uencing the physiological characters. ity in the present study, as the StD-related increasing But in a long period of time, the biomass grew consid- trend was maintained throughout the experimental erablyand aggravated the in£uence of turtle number. process (unpublished data), suggesting that the ACP Our experiment lasted1month and this period is very activity was enhanced by crowding in the StD range short for biomass growth. Hence, it was not the mass set up in the study. of the turtle but the number of turtles in the contain- The serum lysozyme contributed considerably to er that caused the responses. Zhao (2001) reported bacteriolyitc activity, which plays an important role that in turtle farms, juvenile turtles of 50^250 g of a non-speci¢c defence mechanism in ¢sh (Moyner, could be reared at stocking densities as high as 20 Road, Sevatdal & Heum1993).The bacteriolytic activ- ind m 2. This estimate was empirically derived from ity is directly related to the lysozyme activity (Taylor, experience of culturists and the laboratory results of 1983). In ¢sh, crowding stress exhibited varied e¡ects this study do not support it for the negative correla- on lysozyme activity (Rotllant et al.1997;Montero tion between growth and stocking density. The de- et al.1999;Wanget al. 2004). In the present study, we creased growth in crowding groups could be partly observed signi¢cant positive correlations between ly- due to the increasing movement observed, for at a sozyme and stocking density.These ¢ndings suggest higher stocking density more food energy was re- that crowding may enhance lysozyme production or quired for behavioural interactions and thus diverted activity in soft-shelled turtles. Similar results were from somatic growth. For this reason, further study found in gilthead seabream, S. aurata juveniles should be carried out to examine the behavioural as- (Montero et al. 1999), and red porgy, Pagrus pagrus pects associated with crowding, and an appropriate (Rotllant et al. 1997), subjected to chronic crowding stocking density should be sought to balance the po- stress. The non-signi¢cant di¡erences in the splenic tential increasing net production and decreasing indices among all treatment groups indicated that growth rate caused by stocking density in turtle the atrophy of lymphoid tissue did not occur in farms. stressed groups. Phagocytic activity was chosen as a non-speci¢c The present results showed a signi¢cant enhance- cellular immune parameter because it is important ment in cellular and humoral-induced non-speci¢c for host defences and has been regarded as a suitable immunity and no impairment in immune tissue. indicator of immunocompetence in stress studies Thus, it is apparent that immunodepression that had (Yin et al. 1995). Our result showed that medium StD been largely described in ¢sh species after crowding signi¢cantly increased the blood phagocytic activity stress (Rotllant et al.1997;Monteroet al.1999;Iguchi compared with the control, but a high StD treatment et al. 2003) did not occur in the soft-shelled turtle. A had no such e¡ect. A previous studyhas reported that possible reason for this di¡erence could be due to stress can either enhance or deplete the phagocytic more e¡ective immune defences in reptiles than in activity (Pyne 1994; Raidal, Love, Bailey & Rose ¢sh. However, this up-regulation of general immu- 2000). The di¡erent responses depended on the dura- nity depletes resources and comes at a metabolic cost, tion and intensity of the stress. Although the me- seen as a reduced total plasma protein load in high chanism of blood cells’ phagocytosis is poorly StD groups, which has been regarded as an adequate understood, the elevated phagocytic activity indi- indicator of physiological metabolic condition in ver- cated enhanced pathogen resistance and clearance. tebrates (Harms, Lewbart, Beasley, Stamper, Chittick The serum haemolytic activity induced by theACP & Trogdon 2002). Previous studies had suggested is a very important non-speci¢c defense mechanism that the reduction may be caused by protein metabo- in turtles, because its activity is much higher in tur- lization to satisfy more energy needs under stressful tles than in mammals (Matsuyama,Tanaka, Nakao & circumstances (Mazeaud, Mazeaud & Donaldson Yano1988). Studies had revealed that theACPactivity 1977;Vijayan, Ballantyne & Leatherland1990). Loch- of gilthead seabream, Sparus aurata L., was ¢rst de- miller and Deerenberg (2000) had reported that in- pleted by chronic crowding stress (Tort, Sunyer, creased energy dedicated to immune responses, Go¤mez & Molinero 1996), and then recovered several although likelyadaptive in the short term for increas- days later. During the recovery courses, haemolytic ing disease resistance, may ultimately come at the activity occasionally exhibited higher levels in cost of decreased ¢tness, especially if immune activa- crowding animals than in the control due to tion is prolonged or excessive. The result reported by ‘overcompensation’.However, this £uctuation cannot Mauck, Matson, Philipsborn and Ricklefs (2005) also

r 2007 The Authors 1384 Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386 Aquaculture Research, 2007, 38, 1380^1386 Stocking density e¡ects on immunity of soft-shelled turtle X Chen et al. demonstrated an inverse relationship between the Gross W.B. & Siegal H.S. (1983) Evaluation of heterophil/ growth rate and the development of components in lymphocyte ratio as a measure of stress in chickens. Avian avian immune system. Along with the reduced SGR, Diseases 27, 972^979. we can conclude that the enhancement of non-speci- Harms C, Lewbart G, BeasleyJ, StamperA, Chittick B & Trog- ¢c immune function in the present study is energy don M. (2002) Clinical implications of hematology and plasma biochemistry values for loggerhead sea consuming and may decrease healthy status in the turtles undergoing rehabilitation. In: Proceedings of the long term. In conclusion, the present results indicate Twentieth Annual Symposium on Biology and that moderate crowding may stimulate the non-spe- Conservation. 29 February^4 March 2000, Orlando, FL ci¢c immune system rather than suppress it. How- (ed. by A. Mosier, A. Foley & B. Brost. NOAA Technical ever, the up-regulation is at the cost of energy Memorandum NMFS-SEFSC-477, pp. 190^191. U.S. De- expenditure and may decrease other aspects of phy- partment of Commerce, National Oceanic and Atmo- sical ¢tness such as growth. Further study should be spheric Administration, Southeast Fisheries Science carried out to investigate the more longer-term e¡ect Center of the National Marine Fisheries Service, Miami, of crowding stress. FL, USA. Iguchi K., Ogawa K., Nagae M. & Ito F. (2003) The in£uence of rearing density on stress response and disease suscept- ibility of ayu (Plecoglossus altivelis). Aquaculture 220, Acknowledgments 515^523. We thank Ms Huang Chenxi, Dr Zhou Xianqing and Jurd R.D. (1994) Reptiles and birds. In: Immunology, A Com- parative Approach (ed. by R.J. Turner), pp. 137^172. Wiley, our colleagues at the Ministry of Education Key NewYork, NY, USA. Laboratory for Biodiversity Science and Ecological Lochmiller R.L. & Deerenberg C. (2000) Trade-o¡s in evolu- Engineering for their helpful suggestions and techni- tionary immunology: just what is the cost of immunity? cal assistances. Financial support was provided by Oikos 88,87^98. the National Natural Science Foundation of China Matsuyama H.,Tanaka K., Nakao M. & Yano T. (1988) Char- (No. 30271014). acterization of the alternative complement pathway of carp. Comparative Immunology12,403^408. Mauck R.A., Matson K.D., Philipsborn J. & Ricklefs R.E. (2005) Increase in the constitutive innate humoral im- References mune system in Leach’sStorm-Petrel (Oceanodroma leucor- Aguirre A.A., Balazs G.H., Spraker T.R. & Gross T.S. (1995) hoa) chicks is negatively correlated with growth rate. Adrenal and hematological responses to stress in juvenile Functional Ecology19,1001^1007. green turtles (Chelonia mydas) with and without ¢bropa- Mazeaud M.M., Mazeaud F. & Donaldson E.M. (1977) Pri- pillomas. Physiological Zoology 68,831^854. mary and secondary e¡ects of stress in ¢sh: some new AlexanderJ.B. & Ingram G.A. (1992) Noncellular nonspeci¢c datawith a general view.Transactions of theAmerican Fish- defence mechanisms of ¢sh. In: Annual Review of Fish Dis- eries Society106,201^212. eases, Vol. 2 (ed by M. Faisal & F.M. Hetrick) pp. 223^247. Montero D., Marrero M., Izquierdo M.S., Robaina L.,Vergara Pergamon Press, NewYork, NY, USA. J.M. & Tort L. (1999) E¡ect of vitamin E and C dietarysup- Bradford M.M. (1976) A rapid and sensitive method for the plementation on some immune parameters of gilthead quantitation of microgram quantities of protein utilizing seabream (Sparus aurata) juveniles subjected to crowding the principle of protein-dye binding. Analytical Biochemis- stress. Aquaculture171,269^278. try 72,248^254. Moyner R., Road K.H., Sevatdal S. & Heum M. (1993) Change Burns C.W. (2000) Crowding-induced changes in growth, in non-speci¢c immune parameters in Atlantic saln, reproduction and morphology of Daphnia. Freshwater Salmo salar L.,induced by Aeromonas salmonicida infec- Biology 43,19^29. tion. Fish and shell¢sh Immunology 3,253^265. Canario A.V.M., Condeca J., Power D.M. & Ingleton P.M. Nephew B.C. & Romero L.M. (2003) Behavioral, physio- (1998) The e¡ect of stocking density on growth in the gilt- logical, and endocrine responses of starlings to head sea-bream, Sparus aurata L.. Aquaculture Research acute increases in density. Hormones and Behavior 44, 29, 177 ^ 181. 222^232. Dash M.C. & Hota A.K. (1980) Density e¡ect on the survival, Pyne D.B. (1994) Regulation of neutrophil function during growth rate and metamorphosis of Rana tigrina tadpoles. exercise. Sports Medicine17, 245^258. Ecology 61,1025^1028. RaidalS.L.,LoveD.N.,BaileyG.D.&RoseR.J.(2000)E¡ectof Foss A., Siikavuopio S.I., Saeher B.S. & Evensen T.H. (2004) single bouts of moderate and high intensity exercise and E¡ect of chronic ammonia exposure on growth in juve- training on equine peripheral blood neutrophil function. nile Atlantic cod. Aquaculture 237,179^189. Research ofVeterinary Science 68,141^146.

r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386 1385 Stocking density e¡ects on immunity of soft-shelled turtle XChenet al. Aquaculture Research, 2007, 38, 1380^ 1386

Rotllant J., Pavlids M. & Kentouri M. (1997) Non-speci¢c agglutinating activity in the gilthead sea bream Sparus immune system responses in the red porgy Pagrus pagrus aurata. Veterinary Immunology & Immunopathology 51, after crowding stress. Aquaculture156,279^290. 179 ^ 188. ShenZ.,ZhangZ.,QuanD.,LiuW.&YinW.(2003)Studieson VanWeerd J.H. & Komen J. (1998) The e¡ect of chronic stress the humoral immune response ofTrionyx sinensis toAero- on growth in ¢sh: a critical appraisal. Comparative Bio- monas sobria inactivated vaccine Acta. Hydrobiology Sini- chemitry and Physiology120A,107^112. ca 27, 27^30. Vijayan M.M., Ballantyne J.S. & Leatherland J.F. (1990) Stone D.A.J., Allan G.L. & Anderson A.J. (2003) Carbohy- High stocking density alters the energy metabolism drate utilization by juvenile silver perch, Bidyanus of brook charr Salvelinus fontinalis. Aquaculture 88, bidyanus (Mitchell).I. Uptake and clearance of monosac- 371 ^ 381. charides following intraperitoneal injection. Aquaculture WangW.,WangJ., Li A. & Cai Z. (2004) The e¡ect of crowding Research 34,97^107. stress on non-speci¢c immune functions of Ctenopharyn- Robert A.W. (1998) Elevated Water Temperature, crowding, godon idellus. Journal of Fishery Sciences of China 11, and food deprivation accelerate ¢n erosion in juvenile 408^412. steelhead. The Progressive Fish-Culturist 60,192^199. WorkT.M., Rameyer R.A., Balazs G.H., Cray C. & Chang S.P. Sunyer J.O. & Tort L. (1995) Natural hemolytic and bacterici- (2001) Immune status of free-ranging green turtles with dal activities of sea bream Sparus aurata serum are ef- ¢bro-papillomatosis from Hawaii. JournalWildlife Diseases fected by the alternative complement pathway.Vet e ri n a r y 37,574^581. Immunolology and Immunopathology 45,333^345. Yin Z., Lam T.J. & Sin Y.M. (1995) The e¡ects of crowding Suomalainen L.R.,Tiirola M.A. & Valtonen E.T. (2005) In£u- stress on the non-speci¢c immune response in fancy carp ence of rearing conditions on Flavobacterium columnare Cyprinus carpio L. Fish and Shell¢sh Immunology 5, infection of rainbow trout, Oncorhynchus mykiss (Wal- 519 ^ 529. baum). Journal of Fish Diseases 28,271^277. Zhao C. (2001) The scienti¢c establishment of Chinese soft- Taylor P.W. (1983) Bactericidal and bacteriolytic activity of shelled turtle rearing density. Scienti¢c Fish Farming 7,9. serum against gram-negative bacteria. Microbiology and Zhou X., Niu C., Sun R. & Li Q. (2002) The e¡ect of vitamin C Molecular Biology Reviews 47,46^83. on the non-speci¢c immune response of the juvenile soft- Tort L., Sunyer J.O., Go¤mez E. & MolineroA. (1996) Crowding shelled turtle, sinensis. Comparative Biochemistry stress induces changes in serum haemolytic and and Physiology131A,917^922.

r 2007 The Authors 1386 Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1380 ^ 1386