Effect of Salinity on the Biosynthesis of Amines in Litopenaeus vannamei and the Expression ofGill Related Ion Transporter Genes

PAN Luqing,LIU Hongyu,and ZHAO Qun

The Key Laboratory of Mariculture of Ministry of Education,Ocean University of China,Qingdao 266003,P.R.China(Received July 11,2012; revised October 21,2012; accepted July 16,2013)

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

1 Introduction

Salinity is an important environmental factor that directly affects the survival,growth,and physiological function of shrimp.The shrimp cultured in saline water are often confronted with sudden salinity change in the breeding process,and sometimes show growth inhibition as well as a low survival(Kumlu,2000; Pan,2007; Freire,2008).It is notable that the physiological adaptation of shrimp acclimated to different salinities is directly related to the process of osmoregulation(Matthew,1975).

Many studies have shown that salinity stress can stimulate the change in nervous endocrine(Sommer,1991; Babili,1996; Kazuyuki,2007),thereby inducing the change in physiological function of a target organ in response to the stress(Pequeux,1995; Morris,2001).Zatta(1987)found that whenCarcinusMaenaswas transferred from 100% to 50% seawater,its dopamine(DA)concentration in hemolymph increased rapidly while both noradrenaline(NE)and 5-hydroxytryptamine(5-HT)concentration declined.Morris and Edwards(1995)confirmed that when injected with DA or cyclic adenosine monophosphate(cAMP),Leptograpsus variegatesexperienced a fast increase in the Na+/K+-ATPase(NKA)activity.This indicated that biogenic amines play a role in stimulating the Na+/K+pump,i.e.,NKA,in the gill ofL.variegates.In marine crustacean,gill is the primary organ for salt transport.Previous studies showed that several regulatory molecules,including DA and cAMP,are involved in the osmoregulation of marine crab gill(Trausch,1989; Mo,2003).Crustacean hyperglycemic hormone(CHH),a component of sinus gland,can increase the Na+transport flux and NKA activity(Spanings-Pierrot,2000).Hence,further studies are needed to study the function model of nervous endocrine factors on NKA and other ion transport when salinity changes.

At present,it remains controversy whether the response of ion transport proteins to salinity stress is resulted from the regulation of preexisting enzymes or the upregulation of relevant gene transcription and mRNA translation.Towle(2001)demonstrated that when the salinity changed from 35 to 5,the blue crabCallinectes sapidushad minor change in the expression of NKA α-subunit and relevant protein gene in the gill.This suggested that the change in NKA enzyme activity as the hyper-smogulatory response is likely resulted from post-translational regulatory process.However,Carlos(2005)reported that the abundance of mRNAs that encode V-AT-Pase β-subunit increased in the euryhaline crabChasmagnathus granulatuswhen it was transferred either from isosmotic salinity(30)to the diluted(2)or the concentrated(45)seawater.The abundance of NKA α-subunit mRNA responded to external salinity strongly.These findings indicated that a complex pattern of transcriptional regulation is caused by salinity change and is related to the conformation of gill.At present,little is known about the physiological adaptation and relevant molecular mechanisms(e.g.,gene expression pattern)of euryhaline crustacean,especially shrimp,in response to salinity acclimation.

The white shrimp,Litopenaeus vannamei,is of a great economic value and has excellent properties in breeding.It can tolerate salinity as low as 1(Saoud,2003),and has recently become one of the main aquatic animals cultured in the coastal region of China.In this study,we characterized the mRNA abundance of NKA α-subunit and vacuolar-type H/-ATPase(V-ATPase)β-subunit in the gill ofL.vannameiby semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)assay,and further evaluated the relationship between relevant protein richness and osmoregulatory adaptation by western blotting and immunocytochemical assays.Results were used to elucidate the molecular mechanism(s)of osmoregulatory adaption and associated regulative effect of biogenic amines in shrimp.

2 Materials and Methods

2.1 Animal

Adult shrimp ofL.vannameiwith an average body length of 9.5 cm ± 0.5 cm were obtained from a commercial farm in Shazikou,Qingdao,China.The shrimp were acclimated in tank(40 cm×50 cm×60 cm)containing aerated water(pH 8.1)with an air-lift at 24℃ ± 0.5℃.Prior to experiment,the shrimp were transferred from salinity 30 to 26 by a decrease of 1 per day,and then acclimated for 2 weeks.Apparently healthy animal at the intermolt stage were chosen for the following experiment.The molt stage was discerned by observing partial retraction of the epidermis in uropoda(Robsertson,1987).Half of the tank water was renewed twice daily.During the acclimation period,the shrimp were fed with a formulated commercial diet daily(Haiyue Company,Qingdao).

2.2 Experimental Design

At the beginning of the experiment,no significant difference was observed in the shrimp weight among different treatments.During the experimental period,water condition was kept the same as that for the acclimation(24℃ ± 0.5℃; pH 8.1 ± 0.1).The salinity grades(21,26,and 31)were achieved by mixing seawater with insolated tap water.The shrimp were transferred from the salinity 26(essentially isosmotic to the hemolymph,Gong,2004)to 21 and 31,respectively,and then sacrificed rapidly at 7 time points(0 h,6 h,12 h,24 h,72 h,144 h and 216 h).Each salinity treatment was prepared in triplicate and 6 shrimp per replicate were sampled at each time point.In addition,10 untreated shrimp were used as the control.

2.3 Expression of NKA α-Subunit and V-ATPase β-Subunit Gene and Their Activity

After sampling,the gill was dissected rapidly from the shrimp and placed in 10 volumes of ice-cold homogenization buffer(250 mmol L?1sucrose,6 mmol L?1EDTANa2,10 mmol L?1Tris,and 0.1% deoxychoic Na,pH 7.5).The diced gill was homogenized at 10000 r min?1for 5 min in a high-speed homogenizer.Total RNA was extracted from the gill using Trizol reagent(Promega,USA).The RNA extract was qualified by running agarose gel and quantified with a Ultrospec 2100 Pro UV/Vis spectrophotometer(Amersham,Swedan,OD ratio=A260:A280)(Panet al.,2007; Liuet al.,2009).

2.4 Western-Blotting and Immunolocalization Assay of NKA α-Subunit

A mouse monoclonal antibody against the NKAT α5-subunit was purchased from the Development Studies Hybridoma Bank(Iowa,USA).For western blotting,the gill was homogenized in a mixture of homogenization medium(200 mmol L?1sucrose,5 mmol L?1EDTA-Na2,10 mmol L?1Tris,1 mmol L?1DTT,and 0.1% deoxycholate Na; pH7.5)and protease inhibitor(10 mg antipain,5 mg leupeptin,and 50 mg benzamidine dissolved,v/v= 100:1).In addition,part of the gill was cut and fixed in 4% paraformaldehyde fixative for 24 h.The fixed specimens were fully dehydrated with ethanol,embedded in paraffin,cut into 7-mm-thick serial sections in parallel to the long axis of the filament,mounted on glass microslides,coated with poly-L-lysine,and immunocytochemically stained as previously described(Liuet al.,2009).

2.5 Extraction and Determination of Hemolymph and Gill Biogenic Amines

2.5.1 Determination of biogenic amine content

The content of biogenic amines was determined using a LS-55 spectrofluorimeter(Perkin-Elmer,USA)in accordance with the fluorescent spectral photometric method(Zatta,1987)with slight modifications.All measurements were done with Milli-Q water(Millipore Corporation,USA).The standards of DA and NE(Sigma,USA)were diluted to 1 μg mL?1and 5-HT to 2 μg mL?1with 0.01 mol L?1NaOH before use.Other prepared reagents included acidity butylalcohol,0.0l mol L?1EDTA,0.5 mol L?1iodine solution,0.7 mol L?1alkalescence sodium sulfite(Na2SO3),5 mol L?1glacial acetic acid(HAC),0.08 mol L?1L-cysteine,0.9 μmol L?1NaIO4,0.7 mmol L?1o-phthalaldehyde,0.067 mol L?1phosphate buffered saline(PBS,pH 7.2),and 0.05 mol L?1boracic acid buffer solution(pH 9).

2.5.2 Extraction and determination of hemolymph biogenic amines

One milliliter of 10-fold diluted hemolymph was sequentially mixed with 2 mL of acidity butylalcohol,0.5 mL of boracic acid buffer solution,and 10 mg of NaCl.The mixture was oscillated for 30 s,and then centrifuged at 3000 r min?1and 0–2℃ for 10 min.The supernatant was collected and mixed with 0.8 mL of 0.1 mol L?1hydrochloride.After oscillated for 30 s and centrifuged at 3000 r min?1and 0–2℃ for 10 min,the substrate-water phase was divided into two parts equally(0.6 mL each).

In a clean test tube,0.4 mL of Milli-Q water was mixed with 2.3 mL of 0.01 mol L?1EDTA solution and 0.1 mL of 0.5 mol L?1iodine solution.The mixture was allowed to sit for 2 min,and then added with 0.2 mL of 0.7 mol L?1alkalescence Na2SO3solution.After another 4 min,0.2 mL of 5 mol L?1HAC was added to the mixture.Thereafter,the tube was incubated in an 80℃ water bath for 5 min,and then cooled down.The concentration of NE was determined by measuring the fluorescence intensity at 385 nm excitation and at 480 nm emission.Then,the solution analyte was recovered,and 0.1 mL of 7.25 mol L?1phosphate solution was added.The mixture was stirred and heated in a boiling water bath for 15 min,and then cooled down.The concentration of DA was determined by reading the fluorescence intensity at 325 nm excitation and at 375 nm emission.

In a second test tube,0.4 mL Milli-Q water was mixed with 0.1 mL of 0.08 mol L?1cysteine solution,0.1 mL of 0.9 μmol L?1sodium periodate solution,2 mL of hydrochlorid,and 0.1 mL of 0.7 mmol L?1o-phthalaldehyde solution.The mixture was heated in a boiling water bath for 10 min,and then cooled down.The concentration of 5-HT was determined by measuring the fluorescence intensity at 365 nm excitation and at 480 nm emission.

2.5.3 Extraction and determination of gill biogenic amines

The gill was homogenized in 4 mL of ice-cold acidity butylalcohol homogenization buffer solution at 10000

r min?1for 5 min.The homogenate was oscillated for 5 min,and then centrifuged at 3000 r min?1and 0–2℃ for 5 min.Two and a half milliliters of the supernatant was transferred to a centrifugal tube containing 5 mL of n-heptane and 0.1 mol L?1hydrochloride.The mixture was centrifuged again under the same conditions.The substratewater phase was equally divided into two parts(0.6 mL each).The remaining procedure was the same as described above.

2.5.4 Determination of the standards and blank control

One blank tube was filled with 1 mL of Milli-Q water,and another three tubes filled with 0.1 mL of standard solutions of DA,NE,and 5-HT,respectively.The blank and standard samples were prepared and tested simultaneously.

The concentration of hemolymph biogenic amines were calculated according to the formula

and that of gill biogenic amines were calculated according to the formula

whereF(t),F(b)andF(s)are the fluorescence intensities of the test sample,blank and standard,respectively.

2.5.5 Statistical analysis

Data were presented as mean ± SD.Statistical comparison of experimental data was performed by one-way analysis of variance(ANOVA)and Duncan’s multiple range test in SPSS 11.0.The level of statistical significance was set atP<0.05.

3 Results

NKA α-subunit and V-ATPase β-subunit mRNA abundance was significantly influenced by salinity in gill ofL.vannamei(P<0.05),but not in control(P>0.05).At salinity 21,the NKT mRNA abundance peaked at 24 h and remained at a relatively high level afterwards.At salinity 31,the NKT mRNA abundance slowly declined to a slightly lower level compared with that of the control(P>0.05).Despite the abundance was relatively low,it varied in a similar trend as that of NKA(Fig.1).

Fig.1 Effects of salinity on the expression of Na+/K+-ATPase α-subunit and V-ATPase β-subunit mRNAs in gill of Litopenaeus vannamei(A:Na+/K+-ATPase; and B:VATPase).Data are represented as the mean values of triplicate measurements(bars indicate standard errors).

The salinity had a significant effect on the NKA activity(P<0.05)but not on the V-ATPase activity inL.vannamei(P>0.05).No significant variation was observed in either NKA or V-ATPase activity in the control.During the experiment,the NKA activity clearly varied in timeand dose-dependent manner and stabilized at 72 h when the salinity was 21 and 31.There was a negative correlation between the level of NKA activity and salinity(Fig.2).

Fig.2 Effects of salinity on the enzymatic activities of Na+/K+-ATPase and V-ATPase in gill of Litopenaeus vannamei(A:Na+/K+-ATPase; and B:V-ATPase).Data are represented as the mean values of triplicate measurements(bars indicate standard errors).

Salinity significant influenced the NKA α-subunit gene expression in an evident time-dependent manner.At salinity 21,the NKA α-subunit gene expression level substantially increased when the salinity changed by 5(P<0.05),while at 31,the NKA α-subunit gene expression slowly increased in the first 24 h when the salinity increased by 5,and then declined to the control level(P>0.05).There was no significant change in the NKA α-subunit gene expression level in the control(Fig.3).

After salinity change,the content of NKA α-subunit increased in the gill epithelium ofL.vannameiwhen the salinity changed to 21.Immunolocalization assay showed that the amount of NKA α-subunit on immunoblots gradually increased and peaked at 12 h when the salinity changed to 21,showing a significant time-dependent response(P<0.05); whereas when the salinity changed to 31,the amount of NKA α-subunit increased slowly,and then declined rapidly to the control level(P>0.05).No significant variation was found in the content of NKA α-subunit in the control(Fig.4).

Fig.3 Effects of salinity on Na+/K+-ATPase α-subunit protein expression in gill of Litopenaeus vannamei.All data are represented as the mean values of triplicate measurements(bars indicate standard errors).

Fig.4 Effects of salinity on immunoblots of Na+/K+-ATPase in gill of Litopenaeus vannamei.Data are represented as the mean values of triplicate measurements(bars indicate standard errors).

Salinity had a clear time- and dose-dependent effect on the concentration of hemolymph biogenic amines inL.vannamei,but no significant variation were found in the concentration of hemolymph biogenic amines between the control and salinity 31(Fig.5).When the salinity changed to 21,the 5-HT concentration significantly increased to the highest level at 72 h,and then declined slowly to the control level(Fig.5A); whereas the DA concentration increased within the first 12 h and peaked at 24 h after salinity change,then rapidly decreased to the control level(Fig.5B).

Salinity had a significant effect on the concentration of gill biogenic amines inL.vannameiin different treatments,with no significant changes observed in the control group.When the salinity changed to 21 and 31,the gill 5-HT concentration first declined,and then slowly recovered to the control level from 12 h to 24 h(Fig.6A); whereas the corresponding DA concentration varied in an opposite trend.When the salinity changed to 21,DA concentration increased to the highest at 12 h after salinity change; whereas when the salinity changed to 31,DA concentration slowly declined within the first 12 h and then stabilized at half of the initial level(Fig.6B).

Fig.5 Effects of salinity on biogenic amine concentration of hemolymph in Litopenaeus vannamei(A:5-HT; and B:DA).All data are represented as the mean values of triplicate measurements(bars indicate standard errors).

Fig.6 Effects of salinity on biogenic amine concentration of gill in Litopenaeus vannamei(A:5-HT; and B:DA).All data are represented as the mean values of triplicate measurements(bars indicate standard errors).

4 Discussion

NKA of branchial epithelium is located at the basal membrane of most epithelial cells and acts as a key ion transport enzyme for osmoregulation of euryhaline crustacean(Pequeux,1995).It directly mediates the transfer of Na+/K+and affects other apical and basolateral transporters such as the Na+/H+exchanger(Towle,1997),V-type H+-ATPase(Weihrauch,2002),and Na+/K+/2Cl?co-transporter(Towle,1997).Euryhaline crustacean may utilize the branchia apical membrane to exchange Na+/H+and Cl?/HCO3?,as powered by a basal membrane NKA.

4.1 Effect of Salinity on NKA Activity in L.vannamei

Many studies showed that the gill epithelium NKA activity substantially decreased in crustacean when exposed to low salinity,e.g.,Callinectes sapidus,Homarus gammarus(Lucu and Devescovi,1999)andChasmognathus granulate(Castiho,2001),indicating that the NKA activity has a negative correlation with salinity.In the present study,the NKA activity substantially decreased after sudden salinity change(26 to 31)and only stabilized after 72 h,which is similar to the result described by Castiho(2001).However,there was no significant difference in the corresponding V-ATPase activity.This result was consistent with the previous finding of Weihrauch(2002)that V-ATPase activity ofCarcinus maenasdid not change with salinity.Taken together,these findings suggested that NKA is the main component involved in osmoregulation under salinity change,and that the osmoregulation has an obvious time-dependent pattern.In the present study,the NKA activity was suppressed by high salinity and activated by low salinity in gill epithelium of euryhaline osmoregulatory crustacean.This observation suggested that crustacean osmoregulators are hyperosmotic in fresh water or diluted seawater,and need to keep the gill NKA activity at a high level in order to absorb enough Na+for compensation of the Na+loss(Pan,2007).In high-salinity media,the osmotic pressure and Na+concentration of hemolymph are close or equal to the surrounding environment.Thus,the excretive and diffused Na+will be reduced and the activity of gill NKA will decline.Previous work showed that in the apical membrane of gill cells,V-ATPase assisted NKA to absorb Na+when the crustacean was acclimatized to the extremely low salinity of the surrounding environment(Onken and Riestenpatt,1998).However,we observed no obvious variation in the V-ATPase activity,possibly due to the effect of the hyperosmotic environment(Riestenpatt,1995; Onken and Putzenlechner,1995)

4.2 Effect of Salinity on the Abundance of NKA Gene Transcripts in L.vannamei

Despite substantial research of NKA activity in crustacean gill associated with salinity change,it remains unclear whether the increase in NKA activity is resulted fromde novosynthesis of NKA mRNA or post-translational processes such as subunit assembly,membrane trafficking,or cell signaling(Carlos,2005).PCR amplification and sequence analysis of NKA α-subunit gene cDNA to some extent contributed to understanding of the variation in NKA enzymatic activity(Towle,2001).In the present study,results showed that sudden salinity change possibly induced the synthesis of NKA α-subunit mRNA.After the shrimp were transferred from salinity 26 to 21 for 24 h,the abundance of gill NKA α-subunit mRNA markedly increased,suggesting that the expression of NKA subunit gene is the primary molecular mechanism that determines the increase of NKA activity.After salinity change from 26 to 31,the abundance of NKA α-subunit mRNA was found to decline slowly and slightly.This observation indicated the activity of NKA was largely restrained,and the encoding post-translation was interrupted.Moreover,the NKA α-subunit mRNA synthesis lagged behind the enzymatic activity change,likely reflecting the preliminary response of preexisting enzymes to the salinity change.The transfer to a hypoosmotic environment may also be a stimulus for the increase in the NKA α-subunit mRNA abundance.In addition,immunolocalization analysis indicated that before and after the salinity change,there were significant changes in the NKA α-subunit gene expression in correspondence to the changes in protein level.The different expression of NKA and V-ATPase subunit gene indicated that NKA is the main mediator in salinity acclimation.The V-ATPase likely takes part in the regulation of extreme variation in ambient salinity(Carlos,2005).Future studies are needed to study the possible mechanisms such as expression and translational control,as well as the synthesis of NKA α-subunit.

4.3 Effect of Salinity on the Biogenic Amines in L.vannamei

Biogenic amines,which transduce signals as neuroregulators,are widely found in the nervous system and peripheral organs of crustacean(Freire,1995; Halperin,2004; Chiu,2006).Spanings-Pierrot(2000)confirmed that DA is involved in osmotic regulation and ion modulation in crustacean.Mo and Trausch(1998)concluded that biogenic amines elevated cAMP concentration by stimulating the adenylate cyclase activity.Then,the cAMP-dependent kinase acts on the target enzyme(e.g.,NKA)to stimulate protein phosphorylation and to finally reach the osmotic balance(Sommer and Mantel,1991).However,a large body of works have been focused on the perfused isolated gill,and little information is available for biogenic amines and NKA control in salinity variation(Tierney,2003).Péqueux(2002)measured the concentration of 5-HT,NE,and DA in gill and haemolymph extracts ofEriocheir sinensis10 days after the crabs were acclimated to diluted seawater.Salinity stress likely activates aquatic hyper-regulators and release neuroamines(e.g.,DA and 5-HT)from the pericardial organs(Lovett,1995),thereby stimulating NKA activity and NaCl uptakeviaa cAMP-mediated phosphorylation process(Morris,2001).

Our results showed that salinity had significant effect on the concentration of biogenic amines in the hemolymph and gill ofL.vannamei,with obvious time- and dose-dependent responses.The concentration of DA and 5-HT in gill of shrimp declined when the salinity changed from 26 to 31.However,after the salinity changed from 26 to 21,the concentration of 5-HT declined while that of DA increased in gill of shrimp.In addition,the DA concentration of gill increased and peaked at 12 h after salinity change from 26 to 21; while the change in DA concentration occurred in hemolymph later and peaked at 24 h after salinity change.The change in 5-HT concentration in hemolymph lagged behind that in DA concentration and peaked at 72 h after salinity change.We deduce that osmotic regulation ofL.vannameiinvolved two steps:1)the short-term regulation relies on induction and stimulation of biogenic amines on the preexisting enzymes,and 2)the long-term regulation includes two pathways:a)the biogenic amine system stimulates the membrane of gill cells to increase the numbers of both NKA sites and ion channels(Trausch,1989),and b)the endocrine factor cAMP stimulates the phosphorylation to synthesize new enzymes(Mo and Trausch,1998).We speculate that in a low osmotic environment,the great loss of ions could stimulate the endocrine system to produce large quantities of DA(Simonetta,2005).The biogenic amine syntherases(e.g.,ornithine decarboxylase)were then stimulated to synthesize biogenic amines,further elevating the cAMP concentration by the stimulation of the adenylate cyclase activity.The cAMP-dependent kinase acts on the target enzyme(NKA)to stimulate the phosphorylation of protein to reach the final osmotic balance.Meanwhile,5-HT exerts a long-term regulatory effect on osmotic adaption by stimulation of CHH release.Therefore,this work confirmed that DA and 5-HT play different regulatory roles in the osmotic adaption and modulation of shrimp under environmental salinity change.

Acknowledgements

This study was supported by the National Natural Sciences Foundation of China(31072193)and the Scientific Research Foundation for Outstanding Young Scientists of Shandong Province(2006BS07005).The authors would like to thank the members of the Laboratory of Environmental Physiology of Aquatic Animal,Ocean University of China,for their assistance in sampling.

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