A Comparative Study on the Nonspecific Immunity of Juvenile Litopenaeus vannamei ever Inhabiting Freshwater and Seawater

JIA Xuying,DING Sen,WANG Fang,,and DONG Shuanglin

1) Key Laboratory of Mariculture of Ministry of Education of China, Ocean University of China, Qingdao 266003,P.R.China

2) Laboratory of Riverine Ecological Conservation and Technology,Chinese Research Academy of Environmental Sciences,Beijing 100012, P.R.China

? Ocean University of China,Science Press and Spring-Verlag Berlin Heidelberg 2014

1 Introduction

The Pacific white shrimp(Litopenaeus vannamei)is a high-density aquacultured species.It grows fast and tolerates a wide range of salinity,making it one of the most important cultured shrimp species in the world(Wyban and Sweeney,1991).Since the development of its rearing methods over the past 10 years(Jianget al.,2004),it has been widely adopted as a freshwater cultured species in the United States,Thailand and China,especially in southern China and some inland regions(Saoudet al.,2003; Chenget al.,2006; Aranedaet al.,2008).The yield ofL.vannameiin China surpassed 1.2 million tons in 2010,half of which is from inland culture,one of the mainstay industries of China aquaculture(BOF,2011).

As in seawater or brackish shrimp culture,disease is one of the main factors influencing the freshwater shrimp culture yield.The occurrence of disease is closely related to a reduction in the immunity of shrimp（le Moullac and Haffner,2000; Chenget al.,2003).There have been reports that immunity is affected by molt stage(le Moullacet al.,1997; Corteelet al.,2009)and lower salinity(Liet al.,2008; Linet al.,2012)typically varying from 2.5 to 5 practical salinity units in aquaculture.However,there have been no detailed reports on the nonspecific immunity ofL.vannameiinhabiting freshwater(salinity ≤ 0.5)at different molt stages.

The present study evaluated the changing trend of several nonspecific immunity parameters of juvenileL.vannameiinhabiting freshwater among molt stages,and compared these parameters with those of similar shrimp in seawater.The immunity parameters compared included total hemocyte count(THC)and respiratory burst(RB)and the activity of phenoloxidase(PO),nitric oxide synthase(NOS),and lysozyme(LY).

2 Materials and Methods

2.1 Source and Acclimation of Shrimp

JuvenileL.vannameiwere collected from Jiaozhou Shrimp Farm(Qingdao,Shandong Province,China)where salinity was 18–20.Within 2 h,the shrimp were transported to Key Laboratory of Mariculture,Ocean University of China(Qingdao,Shandong,China).Selected healthy shrimp were reared in two fiberglass tanks(150 cm × 150 cm × 100 cm,water salinity 20).

After 7 d,600 shrimp with body lengths of 7–8 cm were divided into freshwater and seawater group,5 tanks(120 cm × 120 cm × 80 cm; 1.0 t water; 60 shrimp)each.The salinity of seawater group was elevated to 30 at a rate of 2–3 d?1by adding natural seawater(salinity 30); while the salinity of freshwater group was reduced to 5 at the same rate by adding freshwater,and then to <0.5 at a rate of 0.5–1.0 per day.Once the salinity reached the designed,shrimp were acclimated for 15 d,deprived of food for 24 h with those in a similar size(wet body weight 3.93 ± 0.41 g)chosen for analysis.

During experiment,shrimp were fed with formulated feed twice a day(at 07:00 and 16:00).The temperature was maintained at 25.0℃ ± 0.5℃ using an air conditioner.Aeration was provided continuously to keep dissolved oxygen above 6.0 mg L?1.The photoperiod was 14L:10D.Every day,approximately 1/3 to 1/2 of the water was exchanged.

2.2 Determination of Molt Stages

An ecdysis cycle is divided into different molt stages:post-molt(A and B),inter-molt(C),and pre-molt(D0,D1,D2,and D3).Using an Olympus microscope(×40),different molt stages were identified and confirmed by comparing setae morphology(Drach,1939; Cesaret al.,2006).

2.3 Sample Collection and Analysis

At each molt stage,20 shrimp from freshwater and seawater,respectively,were selected randomly,10 were used to determining THC,RB,and the activity of NOS and PO,and the other 10 were used to determining the activity of LY.To meet the required sample size in order to perform statistical analyses,every two hemolymph samples were pooled as one.The hemolymph was collected from the abdominal sinusoids of juveniles using a 1-mL sterile syringe containing pre-cooled anticoagulant(10 mmol L?1EDTA Na2,450 mmol L?1sodium chloride,10 mmol L?1potassium chloride,10 mmol L?1HEPES,pH 7.3).The proportion of hemolymph to anticoagulant was 1:2.

A part of each anticoagulant hemolymph sample was directly used to determining THC,and the rest was centrifuged at 3000 r min?1and 4℃ for 10 min.The supernatant was collected in order to measure PO or LY activity; while the precipitants were used to measuring NOS activity and RB.THC was determined by counting with a hemocytometer under an Olympus phase contrast microscope(×40).Hemocyte numbers were calculated as the average of that in two counting chambers of hemocytometer,,and the concentration of hemocytes was calculated using the following formula:THC(mL)= average number of hemocytes in one small cell × 4 × 106× dilution multiple.

Song and Hsieh’s method(1994)was used to determining RB with some modifications.300 μL anticoagulant hemolymph was subjected to centrifugation at 3000 r min?1and 4℃ for 10 min.Plasma was discarded and 100-μL nitrotetrazolium blue chloride(NBT)solution(0.3%)was added and thoroughly mixed at 37℃ for 30 min.The mixed fluid then underwent centrifugation at 3000 r min?1and 4℃ for another 10 min with supernatant discarded.After 600 μL of methanol was added to the sediment,the solution was evenly mixed.Mixture was subjected to centrifugation(3000 r min?1,4℃)for 10 min again with the supernatant discarded.The precipitant was exposed to the air,then potassium hydroxide(KOH)(360 μL,2 mol L?1)and 420 μL methyl sulfoxide(DMSO)were added and treated by thorough mixing.The sample was spotted in a 96-well microplate,and the absorbance rate was determined at a wavelength of 630 nm.

PO activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine(L-DOPA)(Ashida,1971; Hernándezet al.,1996).The sample,consisting of 50 μL hemolymph supernatant and 50 μL trypsin solution(0.1 mg mL?1in CAC buffer,CAC buffer:10 mmol sodium cacodylate,10 mmol CaCl2,pH 7.0),was added to a 96-well microplate and incubated at room temperature for 10 min.Fifty microliters of L-DOPA solution(3 mg mL?1in CAC buffer)was then added to the plate and incubated at room temperature for another 10 min.The 96-well microplate was subsequently placed in an enzyme-labeling instrument(Thermo Labsystem MK3),and the enzyme activity was measured at a wavelength of 492 nm.An enzymatic unit was defined as an absorbance increase of 0.001 at 492 nm per milliliter sample per minute under experimental conditions.

NOS activity was tested using Nitric Oxide Synthase Assay kit(Nanjing Jian-Cheng Bioengineering Institute).NOS catalyzes L-arginine into nitric oxide,which reacts with nucleophilic material,producing a colorful compound.The size-of-extinction coefficient was measured at 530 nm.An enzymatic unit of NOS was defined by the production of 1 nmol nitric oxide per milliliter sample per minute.

LY activity was measured using Lysozyme Assay kit(Nanjing Jian-Cheng Bioengineering Institute).A bacterial suspension ofMicrococcus lysodeikticusfreeze-dried powder was used as substrate or used to prepare the bacterial suspension that was then used as substrate.Both the absorbance of the sample tubes and standard tubes were measured at a wavelength of 530 nm.The LY activity of the sample tube was calculated according to the standard tube concentration.

The concentration of total plasma protein was assayed with Lowry’s method(Lowryet al.,1951).

2.4 Statistical Analysis

The results are presented as mean ± S.E.Data were processed with SPSS 11.5 statistical software.The combined effect of salinity and molt stage on nonspecific immunity was analyzed using a two-way ANOVA.If an interaction between molt stage and salinity was detected,the Student-Newman-Keuls(SNK)test was conducted.If an interaction between molt stage and salinity was not detected,the one-way analysis and SNK test were con-ducted to analyze differences among molt stages within the same water condition,whereas independent-samplest-tests were conducted to analyze differences of global means in both freshwater and seawater groups and differences between freshwater and seawater during the same molt stage.Differences are significant atP< 0.05.

3 Results

3.1 Total Hemocyte Count

The THC ofL.vannameiat different molt stages is illustrated in Fig.1.The THC ofL.vannameiwas significantly affected by salinity and molt stage,and no interaction between salinity and molt stage was found(P= 0.182).In seawater,THC increased gradually over the molt cycle from the lowest at post-molt stages(A and B)(52.5 × 105and 80.9 × 105ind mL?1,respectively),reaching the highest,110.9 × 105ind mL?1,at stage D3.From the lowest at stage A(102.6 × 105ind mL?1)in freshwater,THC increased gradually to the highest at stage D1(130.5 × 105ind mL?1).The THC ofL.vannameiwas significantly higher in freshwater than in seawater at molt stage A,B and D1(P< 0.05).The average of THC at all molt stages was significantly higher in freshwater than in seawater(P= 8.57e?8)(Table 1).

Fig.1 Total hemocyte count of L.vannamei across molt stages.Values are mean ± S.E.* indicates significant differences between seawater and freshwater.Different lowercase letters indicate significant differences among molt stages in seawater,whereas different capital letters indicate significant differences among molt stages in freshwater.

Table 1 The average of immune factors of L. vannamei

3.2 Phenoloxidase Activity

Salinity and molt significantly influenced the PO activity ofL.vannamei(P< 0.05); but the interaction between salinity and molt stage was not significant(P= 0.583).In both seawater and freshwater,the PO activity changed in a similar way over the molt cycle(Fig.2).In seawater,PO activity was 44.2 U min?1and 39.4 U min?1at stage A and B,respectively,which increased to 87.5 U min?1at stage C,and then decreased gradually across the pre-molt stages.In freshwater,PO activity was 31.0 U min?1and 26.0 U min?1at stage A and B,respectively,which increased to 45 U min?1at inter-molt stage; and touched the lowest(22.2 U min?1)at stage D3.PO activity was significantly lower in freshwater than in seawater across all molt stages except for stage A and B(P< 0.05).As shown in Table 1,the average of PO activity of all molt stages was significant lower in freshwater than in seawater(P= 6.31e?9).

Fig.2 Phenoloxidase activity of L.vannamei across molt stages.

3.3 Respiratory Burst

Fig.3 Respiratory burst of L.vannamei across molt stages.

Two-way ANOVA revealed that both molt and salinity significantly affected the RB ofL.vannameiand there was no interaction between them(P= 0.060).In seawater,RB was the lowest at stage A,which then gradually increased to the maximum at stage D3,4.8 times higher than that at stage A.In freshwater,RB initially decreased and then increased gradually from post-molt(stage A and B)to pre-molt(stage D0to D3).The lowest activity was found at stage C; while the highest was found at stage D3.RB was significantly lower in freshwater than in seawater across molt stages except for A and B(P< 0.05)(Fig.3).The average RB of all molt stages was significantly lower in freshwater than in seawater(P= 2.23e?7)(Table 1).

3.4 Nitric Oxide Synthase Activity

NOS activity ofL.vannameiwas significantly affected by salinity and molt although their interaction was not significant(P= 0.653).In seawater,NOS activity remained low at stage B and C(16.2 U mL?1and 16.4 U mL?1,respectively),then increased gradually across pre-molt stages.At stage D1and D3,NOS activity was 20.0 U mL?1and 20.6 U mL?1,respectively.In freshwater,NOS activity increased gradually over the molt cycle.Low NOS activity was recorded at post-molt stages(A and B,13.6 and 10.8 U mL?1,respectively); whereas high NOS activity was found at stage D1and D2(17.3 and 17.9 U mL?1,respectively).At stage B and D3,NOS activity was significantly higher in freshwater than in seawater(P< 0.05)(Fig.4).The average of NOS activity of all molt stages was significantly lower in freshwater than in seawater(P= 1.13e?4)(Table 1).

Fig.4 Nitric oxide synthase activity of L.vannamei across molt stages.

3.5 Lysozyme Activity

Two-way ANOVA indicated that LY activity ofL.vannameiwas significantly affected by salinity; however the interaction between salinity and molt was not significant(P= 0.999).LY activity remained constant throughout the molt cycle in both seawater and freshwater(Fig.5).LY activity between freshwater and seawater was not significantly different across molt stages.The average LY of all molt stages was significantly lower in freshwater than in seawater(P= 0.009)(Table 1).

Fig.5 Lysozyme activity of L.vannamei across molt stages.

4 Discussion

The internal defense(immune)of aquatic animals includes humoral and cellular systems(Qiuet al.,2011).The hemocytes are the major components of cellular immune system in crustaceans.These cells scavenge foreign substances(e.g.,bacteria,fungi,carmine particles)through phagocytosis,encapsulation and cell aggregation(Smith and Ratcliffe,1980),playing an important role in non-specific immune defense.They also synthesize and release immune factors for humoral immunity in crustaceans(Goldenberget al.,1984).Therefore,the THC may be a convenient indicator of stress-induced immune system activity.Temperature and chemical and biological stress can all change the THC(Cheng and Chen,2001; Chenet al.,2007; Xianet al.,2010).Marrec(1944)pointed out that the maximum number of hemocytes appeared at inter-molt stage in crustaceans while mitosis in hematopoietic tissues peaks(Charmantier,1972).Subsequent researches inMarsupenaeus japonicus(Tsinget al.,1989)andL.stylirostris(Le Moullacet al.,1997)revealed that the quantity of hemocytes is actually influenced by molt cycle.In the present study,the THC of the shrimp in seawater was the lowest at stage A and B and increased gradually to the maximum at stage D3.A similar trend was also documented in freshwater.These findings were consistent with those found inM.japonicus(Tsinget al.,1989).After ecdysis,a number of newly matured hemocytes are released into blood vessels(Hoseet al.,1992).Meanwhile,the shrimp’s body also absorbs a great deal of water,effectively reducing the concentration of hemocytes in the blood,thus their immune function is poor.

Previous studies reported that an abrupt drop in salinity could reduce the quantity of hemocytes inMacrobrachium rosenbergiiandL.vannamei.The quantity of hemocytes recovered gradually over time(Cheng and Chen,2000; Wang and Chen, 2005).In the present study,the average THC of the testers was significantly higher in freshwater than in seawater.This contrasting result may be attributed to the specific experimental conditions.Pre-vious studies always focused on abrupt fluctuations in salinity.However,this experiment used a long acclimation period.Therefore,we suggested that the two salinity change patterns have different effects on the quantity of hemocytes.

In the immune system of crustaceans,phagocytes produce toxic substances,including a type of reactive oxygen species,to kill foreign organisms(Muńozet al.,2000).This phenomenon,termed respiratory burst(RB),has also been found in neutrophils and macrophages in mammals.Liuet al.(2004)found a low level of RB inL.vannameiafter ecdysis.Bell and Smith(1993)reported that RB was accompanied by phagocytosis in hyaline cells and the level of RB was closely related to the quantity of hyaline cells.In this study,the RB level was low after ecdysis and then increased gradually in freshwater and seawater,exhibiting a pattern of change similar to that seen in THC.These findings indicated that the hyaline cells account for a large proportion of hemocytes.Undoubtedly,the lower RB after ecdysis increases the susceptibility of shrimp to pathogens.

Phenoloxidase not only participates in the formation of melanin,cuticle hardening,and wound healing,but also plays an important role as a non-self-recognition system in immune defense reactions(Ashida and S?derh?ll,1984).In the present study,the PO activity ofL.vannameiwas the highest at inter-molt stage and the lowest during ecdysis in both seawater and freshwater.Similar results were previously reported inL.stylirostris(le Moullacet al.,1997).S?derh?llet al.(1986)found that the granule cells,which can release pro-PO,contain ribosomes,endoplasmic reticulum,and a large quantity of granules.In the present study,the quantity of hemocytes remained low after ecdysis,leading to a reduction in pro-PO discharge and a drop in phenoloxidase activity.Yehet al.(2009)indicated that the expression of pro-PO-I gene was the highest after ecdysis,and that transcription and translation also occurred at the same time.These activities are forerunners to the sharp increase in PO activity at inter-molt stage.

The cuticles of crustaceans are their first defensive barriers to resist external environmental matter.During the process of calcification that builds the cuticle,quinone transformed by PO interacts with keratin and chitin to produce a horny layer that can resist foreign substances(Qi,2005).Previous research in insects revealed that pro-PO is transferred to the cuticle from the hemolymph by transepithelial transportation(Tsunaki and Masaaki,2001).Meanwhile,the genes controlling the synthesis of pro-PO do not exist in the epithelium beneath the corneum(Ashida and Bery,1995),indicating that pro-PO in the corneum comes from the hemolymph.Stevenson and Adomako(1967)reported that PO existed in the new upper epidermis of crayfish(Orconectes obscures) and decreased rapidly at stage B,accompanied by cuticle hardening.Presumably,the transportation of pro-PO from the hemolymph to the corneum after ecdysis results in low PO level in hemolymph.

The NOS system can produce nitric oxide that has antibacterial properties(Taniaet al.,2008).Recent studies have confirmed that active NOS is plentiful in the histiocytes of aquatic animals,where it apparently kills bacterial pathogens as a non-specific resistance mechanism(Villamilet al.,2002; Garstenet al.,2003).Zhuet al.(2005)reported that changing temperature and salinity had significant effects on NOS activity,and the activity decreased as the change amplitudes of environmental factors became steeper.In the present study,NOS activity inL.vannameiincreased gradually from post-molt to pre-molt in both seawater and freshwater,and the change pattern of NOS activity was similar to that of THC,indicating that there is a close relationship between THC and NOS inL.vannamei.

LY can hydrolyze the acetyl-amino polysaccharides of cell walls of gram-negative bacteria and destroy and remove foreign matter(Liuet al.,1999).The present study showed that the LY level was slightly higher at inter-molt stage,but there were no significant differences in LY level among all molt stages under either seawater or freshwater condition(P> 0.05).LY activity is hardly affected by molt cycle.Therefore,LY may be an important immune factor that is needed to resist pathogen invasion during the molt cycle.

The relationship between immune system parameters in shrimp that were reared at different salinities and their resistance against pathogens has also been reported.Linet al.(2012)reported thatL.vannameithat had been cultured at salinity 2.5 and 5 over a long-term showed a decreased resistance against the bacteriumVibrio alginolyticusand white spot syndrome virus.These individuals also showed much lower RB and activity of PO,SOD and LY than shrimp reared in water with salinities 15,25 and 35.L.vannameishowed decreased RB,reduced activity of PO and SOD,and reduced resistance againstV.alginolyticus24–72 h after transfer from salinity 25 to either 5 or 15(Wang and Chen,2005).These results indicated that shrimp reared at or subjected to low-salinity stress exhibited decreased resistance against pathogens due to reduced immunity.

Compared with that in seawater,the change of immune function among molt stages ofL.vannameiin freshwater was not significant.However,the RB and the activity of PO,NOS and LY were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater(P< 0.05).These findings indicated that wheneverL.vannameiinhabits freshwater,it is likely to be more susceptible to pathogens,and its immune function is different from what it would be in seawater.The functions of cellular immunity ofL.vannameiinhabiting freshwater are enhanced,whereas the functions of humoral immunity are inhibited,resulting in low level of certain immune factors,such as RB and PO activity.

In conclusion,care should be taken when rearing shrimp in freshwater as they have low humoral immune function.Disturbances should be avoided by reducing artificial operations,and external stresses should be eliminated,particularly during the post-molt stage.In addition,it may be worthy to use an immunopotentiator during the post-molt or pre-molt stages in order to maintain immune system function and to reduce the incidence of disease.

Acknowledgements

This work was supported by a project of The Major State Basic Research of China(2009CB118706).

Ashida,M.,1971.Purification and characterization of prophenoloxidase from hemolymph of the silkwormBombyx mori.Archives of Biochemistry and Biophysics,144:749-762.

Ashida,M.,and Soderhall,K.,1984.The prophenoloxidase activating system in crayfish.Comparative Biochemistry and Physiology, 77B:21-26.

Ashida,M.,and Brey,P.T.,1995.Role of the integument in insect defense:Pro-phenol oxidase cascade in the cuticular matrix.Proceedings of the National Academy of Sciences of the United States of America,92:10698-10702.

Araneda,M.,Perez,E.P.,and Gasca-Leyva,E.,2008.White shrimpPenaeus vannameiin freshwater at three densities:Condition state based on length and weight.Aquaculture,283:13-18.

Bell,K.L.,and Smith,V.J.,1993.In vitrosuperoxide production by hyaline cells of the shore crabCarcinus maenas.Developmental and Comparative Immunology, 17:211-219.

BOF(Bureau of Fisheries),2011.China Fisheries Statistic Yearbook 2010.Bureau of Fisheries,Ministry of Agriculture,Beijing,28-30.

Cesar,J.R.O.,Zhao,B.P.,Malecha,S.,Ako,H.,and Yang,J.Z.,2006.Morphological and biochemical changes in the muscle of the marine shrimpLitopenaeus vannameiduring the molt cycle.Aquaculture,261:688-694.

Charmantier,M.,1972.étude préliminaire de la leucopoièse chezPachygrapsus marmoratus(Crustacé,Decapode)au cours du cycle d’intermue.Comptes Rendus de l'Académie des Sciences, 275:683-686.

Chen,M.,Yang,H.,Delaporte,M.,and Zhao,S.,2007.Immune condition ofChlamys farreriin response to acute temperature challenge.Aquaculture,271:479-487.

Cheng,K.M.,Hu,C.Q.,Liu,Y.N.,Zheng,S.X.,and Qi,X.J.,2006.Effects of dietary calcium,phosphorus and calcium/ phosphorus ratio on the growth and tissue mineralization ofLitopenaeus vannameireared in low-salinity water.Aquaculture,251(4):472-483.

Cheng,W.,and Chen,J.C.,2000.Effects of pH,temperature and salinity on immune parameters of the freshwater prawnMacrobrachium resenbergii.Fish and Shellfish Immunology,10:378-391.

Cheng,W.,and Chen,J.C.,2001.Effects of intrinsic and extrinsic factors on the haemocyte profile of the prawn,Macrobrachium rosenbergii.Fish and Shellfish Immunology,11:53-63.

Cheng,W.,Juang,F.M.,Li,J.T.,Lin,M.C.,Liu,C.H.,and Chen,J.C.,2003.The immune response of the giant freshwater prawnMacrobrachium rosenbergiiand its susceptibility toLactococcus garvieaein relation to the moult stage.Aquaculture,218:33-45.

Corteel,M.,Dantas-Lima,J.J.,Wille,M.,Alday-Sanz,V.,Pensaert,M.B.,Sorgeloos,P.,and Nauwynck,H.J.2009.Molt stage and cuticle damage influence white spot syndrome virus immersion infection in penaeid shrimp.Veterinary Microbiology,137:209-216.

Drach,P.,1939.Mue et cycle d’intermue chez les crustacés décapodes.Annales de I’Institut Océanographique,14:103-391.

Garsten,G.K.,Michaela,A.,Christine,L.,Nadja,B.,and Uwe,G.,2003.Reduced expression of the inducible nitric oxide synthase after infection withToxoplasma gondiifacilitates parasite replication in activated murine macrophages.International Journal for Parasitology,33:833-844.

Goldenberg,P.Z.,Huebner,E.,and Greenberg,A.H.,1984.Activation of lobster hemocytes for phagocytosis.Journal of Invertebrate Pathological, 43:77-88.

Hernández,L.J.,Gollas,G.T.,and Vargas,A.F.,1996.Activation of the prophenoloxidase system of the brown shrimpPenaeus californiensis(Holmes).Comparative Biochemistry and Physiology,113C:61-66

Hose,J.E.,Martin,G.G.,Tiu,S.,and McKrell,N.,1992.Patterns of hemocytes production and release throughout the molt cycle in the penaeid shrimpSicyonia ingentis.Biological Bulletin,183:185-199.

Jiang,J.S.,Yang,C.F.,Xue,Y.,and Wang,J.S.,2004.Feeding experiment ofLitopenaeus vannameiin freshwater ponds.Fisheries Science,23:31-32.

Le Moullac,G.,Le Groumellec,M.,Ansquer,D.,Froissard,S.,Levy,P.,and Aquacop,1997.Haematological and phenoloxidase activity changes in the shrimpPenaeus stylirostrisin relation with the moult cycle:Protection against vibriosis.Fish and Shellfish Immunology,7:227-234.

Le Moullac,G.,and Haffner,P.,2000.Environmental factors affecting immune responses in crustacea.Aquaculture,191:121-131.

Li,E.C.,Chen,L.Q.,Zeng,C.,Yu,N.,Xiong,Z.Q.,Chen,X.F.,and Qin,J.G.,2008.Comparison of digestive and antioxidant enzymes activities,haemolymph oxyhemocyanin contents and hepatopancreas histology of white shrimp,Litopenaeus vannamei,at various salinities.Aquaculture,274:80-86.

Lin,Y.C.,Chen,J.C.,Li,C.C.,Morni,W.Z.W.,Suhaili,A.S.N.A.,Kou,Y.Hv,Chang,Y.H.,Chen,L.L.,Tsui,W.C.,Chen,Y.Y.,and Huang,C.L.,2012.Modulation of the innate immune system in white shrimpLitopenaeus vannameifollowing long-term low salinity exposure.Fish and Shellfish Immunology,33:324-331.

Liu,C.H.,Yeh,S.T.,Cheng,S.Y.,and Chen,J.C.,2004.The immune response of the white shrimpLitopenaeus vannameiand its susceptibility toVibrioinfection in relation with the moult cycle.Fish and Shellfish Immunology,16:151-161.

Liu,S.Q.,Jiang,X.L.,Mou,H.J.,Wang,H.M.,and Guan,H.S.,1999.Effects of immunopoiysaccharide on LSZ,ALP,ACP and POD activity ofPenaeus chinensisserum.Oceanologia et Limnologia Sinica,30:278-283.

Lowry,O.H.,Rosebrough,N.J.,Farr,A.L.,and Randall,R.,1951.Protein measurement with the Folin-Phenol reagents.Journal of Biological Chemistry,193:265-275.

Marrec,M.,1944.L’organe lymphocytogène des Crustacés décapodes.Son activité cyclique.Bulletin de L’Institut Océanographique,867:1-4.

Mu?oz,M.,Cede?o,R.,Rodríguez,J.,Van der Knaap,W.P.W.,Mialhe,E.,and Bachère,E.,2000.Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp,Penaeus vannamei.Aquaculture,191:89-107.

Qi,C.Z.,2005.The primary investigation to immunostimulatory fatigue for American white shrimp,Litopenaeus vannameiand Chinese shrimp,Fenneropenaeus chinensis.Master thesis,Ocean University of China.

Qiu,J.,Wang,W.N.,Wang,L.J.,Liu,Y.F.,and Wang,A.L.,2011.Oxidative stress,DNA damage and osmolality in the Paci fi c white shrimp,Litopenaeus vannameiexposed to acute low temperature stress.Comparative Biochemistry and Physiology,Part C,154:36-41.

Saoud,I.S.,Davis,D.A.,and Rouse,D.B.,2003.Suitability studies of inland well waters forLitopenaeus vannameiculture.Aquaculture,217:373-383.

Smith,V.J.,and Ratcliffe,N.A.,1980.Cellular defense reactions of the shore crab,Carcinus maenas:In vivohemocytic and histopahological responses to injected bacteria.Journal of Invertebrate Pathology,35:65-74.

S?derh?ll,K.,Smith,V.J.,and Johansson,M.W.,1986.Exocytosis and uptake of bacteria by isolated haemocyte population of two crustaceans:Evidence for cellular cooperation in the defence reactions of arthropods.Cell and Tissue Research,245:43-49.

Song,H.L.,and Hsieh,Y.T.,1994.Immunostimulation of tiger shrimp(Penaeus monodon)hemocytes for generation of microbicidal substances:Analysis of reactive oxygen species.Developmental and Comparative Immunology,18:201-209.

Stevenson,J.R.,and Adomako,T.Y.,1967.Diphenol oxidase in the crayfish cuticle:Localization and changes in activity during the molting cycle.Journal of Insect Physiology,13:1803-1811.

Tania,R.R.,Georgina,E.,Jorge,H.L.,Teresa,G.G.,Jeannette,M.,Yaisel,B.,Alonso,M.E.,Bécquer,U.,and Alonso,M.,2008.Effects ofEcherichia colilipopolysaccharides and dissolved ammonia on immune response in southern white shrimpLitopenaeus shcmitti.Aquaculture,274:118-125.

Tsing,A.,Arcier,J.M.,and Brehèlin,M.,1989.Haemocytes of penaeids and palaemonid shrimps:Morphology,cytochemistry and hemograms.Journal of Invertebrate Pathology,53:64-77.

Tsunaki,A.,and Masaaki,A.,2001.Cuticular pro-phenoloxidase of the silkworm,Bombyx mori.Journal of Biological Chemistry, 276:11100-11112.

Villamil,L.,Tafalla,C.,Figueras,A.,and Novoa,B.,2002.Evaluation of immunomodulatory effects of lactic acid bacteria in turbot(Scophthalmus maximus).Clinical and Diagnostic Laboratory Immunology, 9:1318-1323.

Wang,L.U.,and Chen,J.C.,2005.The immune response of white shrimpLitopenaeus vannameiand its susceptibility toVibrio alginolyticusat different salinity levels.Fish and Shellfish Immunology, 18:269-278.

Wyban,J.A.,and Sweeney,J.N.,1991.Intensive shrimp production technology:The Oceanic Institute shrimp manual.The Institute,Honolulu,Hawaii,158pp.

Xian,J.A.,Wang,A.L.,Ye,C.X.,Chen,X.D.,and Wang,W.N.,2010.Phagocytic activity,respiratory burst,cytoplasmic free-Ca2+concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp,Penaeus monodonunder acute copper stress.Comparative Biochemistry and Physiology Part C,152:182-188.

Yeh,M.S.,Lai,C.Y.,Liu,C.H.,Kuo,C.M.,and Cheng,W.,2009.A second proPO present in white shrimpLitopenaeus vannameiand expression of the propos during aVibrio alginolyticusinjection,molt stage,and oral sodium alginate ingestion.Fish and Shellfish Immunology,26:49-55.

Zhu,H.Y.,Wang,G.J.,Yu,D.G.,and Xie,J.,2005.Changes in nitric oxide synthase and sensitivity toVibrio parahaemolyticusin serum of white leg shrimp exposed to sudden changes in water salinity.Journal of Zhanjiang Ocean University,25:90-94.