Serum osmolality and ions,and gill Na+/K+-ATPase of spottedtail goby Synechogobius ommaturus(R.)in response to acute salinity changes

Chun Shui,Yonghai Shi,Xueming Hua,Zhonghua Zhang,Haiming Zhang,Genhai Lu,Yongde Xie

aKey Laboratory of Freshwater Fishery Germplasm Resources,Ministry of Agriculture,Shanghai Ocean University,Shanghai 201306,China

bShanghai Fisheries Research Institute,Shanghai 200433,China

1.Introduction

Salinity is an environmental factor that frequently affects the physiology of aquatic organisms(Urbina&Glover,2015).When fish are challenged by salinity change,activities like drinking rate(Evans,Piermarini,&Choe,2005)and osmoregulation(Fielder,Allan,Pepperall,&Pankhurst,2007)change to maintain body osmolality and ionic balance.Euryhalinefish can maintain the ionic composition and osmolality of their internal fl uids relatively constant when environmental salinity changes(Anni et al.,2016).The ability to osmoregulate is essential for their survival in either freshwater or seawater environments.This regulation is achieved by a number of different ionic and osmoregulatory processes.Fish have toadjust and balance the gain and loss of water and serum ion,especially Cl-,Na+,and K+.The change in plasma ion levels are used to monitor the osmoregulatory ability of fish after salinity changes(Stewart et al.,2016).The main mechanism for maintaining serum ion and osmolality balance involves gill Na+/K+-ATPase(NKA)(Stewart et al.,2016;Zhang et al.,2017).This enzyme is located in the branchial chloride cells(Hirose,Kaneko,Naito,&Takei,2003)and can produce a chemical gradient to eliminate excess intra and extra-cellular Na+and Cl-in a hyperosmotic environment and take up Cl-in a hypoosmotic environment(Wood,2011).In most teleosts when salinity changes NKA activity exhibits adaptive changes.The time course of changes in gill NAK activity in response to different environment salinities tends to vary between species.Gill NKA activity in killi fish Fundulus heeroclitus heteroclitus(L.1766)changed 2—3 days after environment salinity increased(Mancera&McCormick,2000),3—7 days were needed for a change in NKA during the breeding migration of coho salmon Oncorhynchus kisutch(Walbaum 1792)(Wilson&Laurent,2002)and in the marine gilthead seabream Sparus aurata L.1758(Laiz-Carrion et al.,2005a,2005b).Osmoregulatory ability of fishes can be evaluated by measuring the NKA activity,serum osmolality and ion levels.

The spottedtail goby Synechogobius ommaturus(Richardson 1862),is a large and demersal fish of the Gobiidae.It is an inshore species of the Asianwestern Pacific Ocean and is widely distributed along the coast of China,Japan and Indonesia.It grows quickly,and has a nearly linear growth curve during the first six months of the first year of its life cycle and then the growth rate slows down,but the body weight and standard length keep on increasing and achieving their maximum by the next April(Wang,Ye,Liu,&Cao,2011,2011b).The species had become a commercial fish in China(Wang et al.,2011).Over fishing and environment pollution pose a threat to this species and has caused sustainability problems for this species.Although in the context of improving sustainability studies about the use of salt marshes(Jin et al.,2007,2010),genetic diversity(Song,Zhang,&Gao,2010)and stock discrimination(Wang et al.,2011a,2011b)of S.ommaturus have been conducted.However,the response of this fish to changes in salinity remain limited and the effects of different salinities on NAK activity,serum osmolality and ion levels have not been performed,despite their relevance for artificial propagation and culture.The present study contributes to better protect the species and improve aquaculture by assessing the effects of acute salinity changes on osmoregulation of S.ommaturus.Such information is important since the habitat of S.ommaturus is frequently affected by uncontrolled salinity fluctuations attributed to evaporation or inundationwith rain.Gill NAK activity,serum osmolality and ion levels are used as indicators of salinity tolerance.

2.Materials and methods

2.1.Animal husbandry and experimental design

Experiments were conducted with spottedtail goby at the facilities of Shanghai Fisheries Research Institute(Shanghai,China).Fish were caught from ponds and maintained in indoor concrete pools prior to experiments.During the 2 weeks of acclimation(salinity 10)and throughout the experiment, fish were fed on commercial feed to apparent satiation once a day(10:00).After acclimation to the experimental conditions,720 fish(65.3±11.8 g)were chosen and randomly allocated between 18 independent tanks(200 L),with 40 juveniles in each tank.Fish were exposed to an ambient photo period of 10L:14D.

Five salinity levels were investigated,namely freshwater,20,30,40 and 50,the culture salinity 10 served as the control.Spottedtail gobies from the different experimental tanks were abruptly and isothermally transferred from the control salinity to the experimental salinities.The effect on the spottedtail gobies of each experimental salinity was determined using 40 fish in triplicate static 200 L tank containing aerated water.

2.2.Sampling

Fish were fasted for 24h prior to sampling.Blood and gill filament samples were taken before transfer to modified salinity and this was the 0 h sample.Three fish were randomly,chosen from each replicate and anesthetized using 100mg/L tricaine methanesulfonate.Fish were sampled at 0.5,1,2,4,8,12,24,48,96,216,360,and 528 h after transfer to the experimental salinity.Blood was collected from the caudal vasculature with 1 mL heparinized syringes,then stored on ice for up to 10h.Serum was obtained by centrifugation at 3700 g for 20 minat 4°C,and the serum was stored at-80°C until analysis.The second and third gill arch was excised and stored at-80°C for the NAK activity test.A sample of water from each replicate was taken at the same time of blood sampling to assess its osmolality and ion content(Table 1).

2.3.Administration of the experiment

Salinity,temperature and pH were measured daily using a YSI model 30 m(Yellow Springs Instruments Inc.,Yellow Springs,Ohio,USA)and YSI model pH 100,respectively.The water temperature(range 11—12°C)and pH(range 8.0—8.6)were measured daily.Dissolved oxygen was maintained at＞80%oxygen saturation throughout the experiment by continuous aeration.Ammonianitrogen and nitrite-nitrogen were measured weekly over the duration of the experiment and had a mean value of＜0.3 mg/L and＜0.1mg/L,respectively.70%of the tank water was exchanged daily.The water salinities tested ranged from 0 to 50 with intervals of 10 and were prepared by adding freshwater or hypersaline seawater to full seawater.The salinity was measured and adjusted by a hand held salinity meter(YSI 30-10,Yellow Spring Instruments,Yellow Springs,OH,USA).

2.4.Biochemical analysis

Serum samples were tested after thawing at 4°C.Osmolality was measured using a cryoscopic osmometer(Gonotec,Osmomat 030,Germany)and reported as mOsm/kg.Serum potassium,sodium and chloride concentrations were tested using an EasyLyte electrolyte analyzer(Medica Corporation,Bedford,MA,USA).Gill NAK activity was determined according to(Zaugg,1982).The NAK speci fic activity was expressed as μmol Pi/(mg protein)·h.

2.5.Statistical analyses

Statistical analyses were performed using SPSS 17.0(SPSS Inc.,Chicago,IL,USA).A One-Way Analysis of Variance(ANOVA)was applied to identify significant differences in serum osmolality and ion concentrations and gill NAK activity in fish challenged with different salinities.If significant differences were detected at the levels of 0.05,then Duncan's multiple tests were used to evaluate the differences among treatments.All data were subject to a test for homogeneity of variances before analysis.The data were plotted using Microsoft Excel(Microsoft,Seattle,WA,USA)and all the values are expressed as mean±standard deviation(M±SD).

3.Results

3.1.Serum osmolality and ions concentration

Serum osmolality and ion concentrations were not significantly different between fish prior to the start of the salinity challenge(0h).No mortality occurred before or immediately after transfer of fish to the salinities 20,30 or freshwater.In contrast,48 h after transfer of fish to 50 salinity all the fish had died and in the fish transferred to 40 salinity all died 360 h later.

Half an hour later after transfer,serum osmolality showed no significant changes(Fig.1a).As time went by serum osmolality increased significantly in fish transferred to the 50 and 40 salinity and peaked at 48 h and 96 h,respectively.Then the osmolality in 40 decreased.The serum osmolality in fish transferred to 20,30 salinity or freshwater was relatively stable except for a slight increase in the 30 group and decrease in the freshwater group(P＞0.05).

Serum Cl-concentration in fish transferred to 20 and 30 salinity remained similar to those of the control fish at 10 salinity(Fig.1b).Fish transferred to 40 and 50 salinity had a significant increase in serum Cl-at 96 and 48 h,respectively relative to the control(P＜0.05).Serum Cl-of fish transferred to freshwater decreased at 4h,then remained significantly lower than that of control fish(P＜0.05).

Table 1 Mean osmolality and ion concentration of treatment water.

Serum Na+showed a similar trend to that of serum Cl-.Relative stability was detected in serum Na+concentration after fish were transferred to 20 and 30 salinity(Fig.1c).The concentration of serum Na+at salinities 40 and 50 increased significantly,and reached its peak at 96 h and 48 h,respectively.In the fish transferred to freshwater,serum Na+decreased at 12h and remained significantly lower than the control group(P＜0.05).

In fish transferred to 50 salinity after 4h the concentration of serum K+increased and peaked at 48h(Fig.1d).There was no significant difference of serum K+among the other treatments(P＞0.05),except that 528 h later,serum K+concentration in fish reared in freshwater was significantly lower than the control group(P＜0.05).

3.2.Gill NAK activity

Half an hour after fish were transferred to 30,40,50 salinity or freshwater,the gill NAK activity increased significantly(P＜0.05)(Fig.2a).Gill NAK activity in fish at 50 salinity increased continually and peaked at 12h,and then decreased but was still higher than the control group(P＜0.05).Similarly,gill NAK activity of fish in 40 salinity reached a peak at 12h and decreased 360 h later(P＜0.05).Regression analysis con firmed the significant effect of salinity on gill NAK activity at 12 h(r2=0.96,P＜0.01)and 360 h(r2=0.77,P＜0.05)(Fig.2c and d).No significant effect of salinity on gill NKA was apparent at 1h(r2=0.07,P＞0.05)(Fig.2b).After 96 h exposure at 20 and30 salinities or freshwater,gill NAK activity fluctuated but was notsigni ficantly different(P＞0.05).528 h later,gill NKA activity of fish in freshwater decreased significantly(P＜0.05)and in fish at20 and 30 salinity were similar to that of control fish.

4.Discussion

To capture the dynamics of change in gill NKA activity,serum osmolality and ion levels of spottedtail goby in response to salinity challenge we carried out a detailed time course study.

No mortality occurred in fish challenged with 20 or 30 salinity or freshwater,which suggests that these fish can tolerate abrupt exposure to a hyper/hypoosmotic environment.The ion concentration in the body fluids changed when fish was challenged by medium salinity fluctuation.The major electrolytes in the body fluids are Na+and Cl-,and both are critical in the osmoregulation(Kaneko,Watanabe,&Lee,2008).During acclimation to a hyperosmotic environment,euryhaline fish undergo two phases: first there is a fast rise in gill-ion fluxes followed by an increase in serum electrolytes and osmolality,this is then followed by a regulatory period in which gill NKA activity increases,functional mitochondria rich cells proliferate,Na+and Cl-efflux increase and serum ion balance is restored(Evans et al.,2005).In the present study,the serum osmolality,Cl-,Na+and K+of spottedtail goby increased in 40 and 50 salinity after transfer indicating that dehydration occurred due to the osmotic efflux of water from the body and excess in flux of ions from the hyperosmotic environment(Hwang,Sun,&Wu,1989).Inversely,serum osmolality,Cl-,Na+and K+of fish transferred directly to freshwater decreased significantly,indicating that osmotic influxoccurred,in accordance with dilution of serum ion concentration even redistribution of ions between serum and cells(Bath&Eddy,1979).However,these changes were transient,the serum osmolality and ion concentration returned to,or was higher than initial levels after 528 h of acclimation.According to(Mylonas et al.,2009),lowered serum Na+and osmolality could be an indication of osmoregulatory difficulties,as previously proposed by(Laiz-Carrion et al.,2005a,2005b).A similar trend was observed in the freshwater group in this study and may indicate that the fish were in osmoregulatory difficulties.Alternatively,it could be the consequence of long term adaptation to the modified environmental conditions and further research is needed to explain the findings of the present study.Relatively stable serum osmolality and electrolyte concentration in fish transferred to 20 and 30 salinities indicated that the spottedtail goby has a strong osmoregulatory ability.

The restoration of homeostasis in fish transferred to a hyperosmotic or hypoosmotic medium is accompanied by changes in gill NKA activity.The change in gill NKA activity plays a pivotal role in adaptation to different salinities(Stewart et al.,2016;Zhang et al.,2017).In the present study,the activity of NKA was significantly affected by changes in salinity.Fish transferred to 40 and 50 salinity had a significant increase in gill NKA activity,that peaked at 12 h and then declined.This could be indicate that the gill Na+/K+-ATPase activity became exhausted and unable to maintain for a prolonged duration the necessary increased activity required to maintain osmoregulation.The regression curve of gill NKA activity showed a significant U-shape with water salinity during the response and adaptionprocesses(Fig.2b),which was in accordance with Jensen et al.(Jensen,Madsen,&Kristiansen,1998)and Laiz-Carrion et al.(Laiz-Carrion et al.,2005a,2005b).It suggested that fish were living in a medium with a salinity equivalent to that of their body fluid(Gaumet,Boeuf,Severe,&Mayergonstan,1995).The change of gill NKA activity as a result of salinity changes has been found largely in euryhaline species.However,results are variable or even con fl icting.Responsiveness of gill NKA activity to salinity altered with species,life stage and experimental conditions and so on.Some reported a positive correlation between salinity and gill NKA activity(Imsland,Gunnarsson,Foss,&Stefansson,2003);however,negative relationship also be detected(Marshall,Emberley,Singer,Bryson,&Mccormick,1999).Further confusion arises since some studies found no effect of salinity on gill NKA activity(Yoshikawa,Mccormick,Young,&Bern,1993).Nevertheless,at the end of this study,no significant difference in gill NKA activity between salinity 20,30 and control groups implied its strong osmoregulatory and highly ion regulation mechanisms.Meanwhile,a decreasing of gill NKA activity obtained in fish transferred to freshwater after 528 h of exposure.One possible explanation is that the primary task of gill NKA activity to excrete excessive NaCl via chloride cells(Whittamore,2012)was exhibited in freshwater since the hypoosmotic environment activated the hypoosmoregulation mechanism for excretion of the excessive water.While maintaining high level of gill NKA activity in freshwater could be energy-intensive,as a matter of course the activity reduced to save more energy for growth and other metabolism activity(Saoud,Kreydiyyeh,Chalfoun,&Fakih,2007).

Fig.1.Serum osmolality(a),chloride(b),sodium(c)and potassium(d)concentration of spottedtail goby(Synechogobius ommaturus)in different salinities.Results are represented as the M±SD.(n=3).The same letter at the same time indicates no significant difference existed between experimental groups.

Fig.2.Gill Na+/K+-ATPase activities(μmol Pi/(mg protein)⋅h)of spottedtail goby(Synechogobius ommaturus)after transfer to different experiment salinities(a)and regression analysis de fined the relationship between the gill NKA activity and salinities after 1 h(b),12h(c)and 360 h(d)transfer.

The results indicatethat spottedtail gobyjuveniles need a period of accommodation for its ion osmoregulatory system to adapt to environment salinity changes.The absence of mortality in spottedtail goby abruptly transferred to a hyper/hypoosmotic environment indicated its strong ion-osmoregulatory ability.All of the data of the present study demonstrated that spottedtail goby can maintain homeostasis across a range of medium salinity changes(salinity 10,20,30 and freshwater).Gill NKA activity played an important role in the restoration of homeostasis and presented a U-shape curve which is the common feature of euryhaline fish.A salinity of 40 appeared to be the upper tolerance limit of spottedtail goby since death occurred after 360 h exposure presumably since the fish failed to acclimate.We recommend salinity below 40 as ultimate salinity tolerance of spottedtail goby in later experiment.The ability of spottedtail goby to tolerate an abrupt salinity change may be an acquired trait since in their natural habitat they are frequently exposed to freshwater intrusion,tidal changes,rainfall and evaporation bringing about rapid alteration of environmental salinity.Understanding the physiological capacity of spottedtail goby to adapt to environment salinity changes is essential information for planning sites for aquaculture.Our results support the idea that the spotted tail goby is suitable for aquaculture in estuaries where rapid fluctuation in salinity occur.Further studies on molecular mechanism will be necessary to better understand the salinity adaptation process.

Acknowledgements

We are very grateful to Xinhua Ye for assistance with blood sampling and tank maintenance.We are also grateful to the anonymous reviewers for their helpful suggestions that improved the manuscript.Financial support was provided by Agriculture Commission of Shanghai(2011/1-4)and Shanghai breed industry development project(2015/17).

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