The Ecophysiological Responses Of Litopenaeus Vannamei Under Freshwater Condition To Environmental Stresses

A series of experiments were conducted in the laboratory to study the effects ofenvironmental stresses (i.e., temperature and non-ionic ammonia) on theeco-physiological characteristics of the shrimps, Litopenaeus vannamei which werecultured in freshwater and seawater. What’s more, the differences of these effectswere analyzed. The main results were listed as below:1. Effects of abrupt change in temperature and non-ion ammonia on survival inL. vannamei under seawater and freshwater conditionsThe effects of temperature and non-ion ammonia stress on survival of L.vannamei under seawater and freshwater condition was studied in this paper. It wasfound that:(1) the interaction among culture condition temperature variation and timeof survival rate in L. vannamei was not significant (P>0.05). When the temperaturechanged from22℃to7,12and37℃suddenly, the survival rate of L. vannameiunder two culture conditions types both demonstrated the downtrend during theextension of changing time. The tolerance ranges of high and low temperaturevariation of L. vannamei under seawater both were10℃，The tolerance ranges of highand low temperature variation of L. vannamei under freshwater were10℃and5℃.The L. vannamei in freshwater had a better adaptive capacity than the ones inseawater in low temperature, while had a worse adaptive capacity in high temperature.(2) The survival rate of L. vannamei was significantly affected by culture condition,temperature variation and exposure time (P<0.05), and the interaction of the threefactors significantly affected the survival rate of L. vannamei (P<0.05). The96hLC50values and―safety level‖of NH3-N were1.612mg·L-1and0.161mg·L-1underseawater condition,0.629mg·L-1and0.063mg·L-1under freshwater condition. The LC50values at12,24,48,72and96h were higher in seawater group than that infreshwater group. The L. vannamei in seawater had a better tolerability to NH3-N thanthe ones in freshwater.2. Effects of temperature stress on ecophysiological characteristics of L.vannamei under freshwater condition2.1Effects of temperature stress on respiratory metabolic enzyme and HSP70of L.vannamei under freshwater conditionAfter temperature stress(16℃←22℃→28℃), changes of activities of hexokinase(HK), pyruvate kinase (PK), lactic dehydrogenase (LDH), succinate dehydrogenase(SDH) and heat shock protein70(HSP70) contents in L. vannamei long-term culturedin freshwater were studied and compared with those in seawater. Results showed that:(1) in the initial stage of acute temperature change, HK, PK and LDH activitiesdifferently increased, while enzymatic activities decreased over time. Except that PKand LDH activities in freshwater shrimps was significantly higher than beforeexperiment, all enzyme activities returned back to the pre-experiment level. Followingtemperature stress, SDH activity in gills and muscles of freshwater and seawatershrimps all decreased first and then increased, and finally reached the level beforeexperiment, while the exception was that SDH activity in gills of seawater shrimpswas significantly lower than that before.(2) Contents of HSP70showed an increase ingills and muscles of freshwater and seawater shrimps after temperature change. Then,it decreased, and returned to the level before experiment in48h. Subjected to lowertemperature stress, shrimps in freshwater and seawater showed a significant change incontents of HSP70in gills (P<0.05), which was different to that in muscles. However,under higher temperature, freshwater shrimps had no significant change in HSP70contents in gills and muscles (P>0.05), while a significant change was observed inseawater shrimps (P<0.05).(3) At a constant temperature of22℃, compared withseawater shrimps, freshwater shrimps had a lower level in HK, PK and SDH activity,and contents of HSP70, but a higher level in LDH activity. 2.2Effects of temperature stress on immunity of L. vannamei under freshwaterconditionAfter temperature stress(16℃←22℃→28℃), changes of total heamocyte counts(THC), phenol oxidase (PO) activity, nitric oxide synthase (NOS) activity, superoxidedismutase (SOD) activity and malondialdehyde (MDA) contents in L. vannameilong-term cultured in freshwater were studied and compared with those in seawater.Results showed that:(1) for shrimps in seawater and freshwater subjected to lowertemperature stress, THC and activity of PO and SOD in hemolymph all significantlydecreased, while NOS activity went back to normal after the initial increase. MDAcontent increased significantly for shrimps in freshwater, but there was no obviouschange in shrimps in seawater. After higher temperature stress, THC in shrimps offreshwater and seawater reached the lowest value in6-12h and then returned back tonormal. In48h, activities of PO and NOS in freshwater shrimps reached the normallevel, while there was a significant decrease in seawater shrimps. SOD activity infreshwater shrimps decreased significantly. But no obvious change was recorded inSOD activity in seawater shrimps. MDA contents in freshwater and seawater shrimpsboth increased initially and then decreased to normal levels.(2) For freshwater andseawater shrimps subjected to lower temperature stress, PO acitivity inhepatopancreas increased first and then decreased, which had a similar tendency toNOS and SOD. MDA contents increased within3-6h after temperature change, andthen decreased gradually to normal levels. After exposure to high temperature,freshwater shrimps showed a similar variation tendency in NOS and SOD activity tothose in lower temperature, while the difference was that no obvious change wasfound in the former. Following temperature increase, seawater shrimps showed asignificant decrease in PO, NOS and SOD activity in hepatopancreas. For freshwatershrimps, MDA content increased to the highest value within6h and got back to thenormal level within48h, which was similar to that in seawater shrimps.(3) Atconstant temperature of22℃, compared to seawater shrimps, freshwater shrimps hada slightly higher THC and significantly higher MDA content, but significantly lower PO, NOS and SOD activities. In general, long-term culture in freshwater would leadto a decreased immunity in Litopenaeus vannamei which suggested a low adaption tolower temperature stress, while it’s supposed to make shrimps more tolerant underhigher temperature stress3. Effects of non-ionic ammonia stress on ecophysiological characteristics of L.vannamei under freshwater condition3.1Effects of non-ionic ammonia stress on respiratory metabolic enzyme and HSP70of L. vannamei under freshwater conditionAfter nonionic ammonia stress(0.1mg·L-1and0.5mg·L-1), changes of activities ofhexokinase (HK), pyruvate kinase (PK), lactic dehydrogenase (LDH), succinatedehydrogenase (SDH), and heat shock protein70(HSP70) contents in L. vannameilong-term cultured in freshwater were studied and compared with those in seawater.Results showed that:(1) nonionic ammonia in lower concentration induced the HKactivity in gills of freshwater shrimps to a significant higher level (P＜0.05), whilenonionic ammonia in higher concentration induced a significant higher level of HKactivity in gills of freshwater and seawater shrimps (P＜0.05). After stress, nosignificant change was observed in HK activity in muscles of freshwater and seawatershrimps (P＞0.05). Following nonionic ammonia stress, an increase was observed inPK activity in gills of all shrimps, and at the end of the experiments, PK activityreturned to the normal level. However, PK activity in muscles showed a differentchange: no significant change was found in seawater shrimps(P＞0.05), which wasdifferent to that in freshwater shrimps. No significant effects of non-ionic ammonia inlower concentration on LDH activity in gills and muscles of freshwater and seawatershrimps were observed (P<0.05). In contrast, nonionic ammonia in higherconcentration imposed a significant effect on LDH activity in gills and muscles offreshwater shrimps (P＞0.05).(2) After ammonia stress, HSP70contents significantlyincreased in freshwater and seawater shrimps, and the former changed moredramatically. In summary, non-ionic ammonia brought a more serious impairment tofreshwater shrimps than seawater shrimps.(3) Following stress, aerobic metabolism in shrimps decreased drastically, but anaerobic metabolism would increased slightly inthe initial period prior to decreasing. It was suggested that non-ionic ammoniaprobably led to a change in main energetic materials in shrimps.3.2Effects of non-ionic ammonia stress on immunity of L. vannamei under freshwaterconditionAfter nonionic ammonia stress(0.1mg·L-1and0.5mg·L-1), changes of totalheamocyte counts (THC), phenol oxidase (PO) activity, nitric oxide synthase (NOS)activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) contentsin L. vannamei long-term cultured in freshwater were studied and compared withthose in seawater. Results showed that:(1) after nonionic ammonia stress, THC andPO activity significantly decreased in freshwater and seawater shrimps(P＜0.05), andthe decreased extent was bigger with the nonionic ammonia concentrationincreasing.(2) Following0.1mg·L-1nonionic ammonia stress the NOS activity offreshwater and seawater shrimps both increased initially and then decreasedsignificantly(P＜0.05),while0.5mg·L-1nonionic ammonia stress, NOS activitydroped sigificantly and rapidly.(3) The activity of SOD in hemolymph changednon-significantly in the initial stage of nonionic ammonia stress, while decreaseddramatically after3-6h (P＜0.05). The SOD activity in hepatopancreas declinedcontinuously after stress.(4) After nonionic ammonia stress, MDA content inhemolymph and hepatopancreas of seawater and freshwater shrimps differentlyincreased. In summary, nonionic ammonia stress weakened the nonspecific immunityand broke the balance of antioxidative system of shrimp. Long-term culture infreshwater would lead to a decreased immunity in L. vannamei which meant thatfreshwater shrimp was more susceptible to nonionic ammonia.4. Effects of temperature and non-ionic ammonia stress on apoptosis of L.vannamei under freshwater conditionEffects of acute temperature change (16℃←22℃→28℃) and non-ionicammonia stress (0.1mg·L-1and0.5mg·L-1) on the cytochrome C (cyt-C) contents andcaspase-3activity in freshwater shrimps under a long-term culture were studied, and they were compared with those in seawater shrimps. Results showed that:(1) after5dof acute temperature change, hemolymph cyt-C contents in freshwater and seawatershrimps were all higher than before (P<0.05). At lower temperature, cyt-C contents inhepatopancreas in freshwater shrimps increased significantly in5d (P<0.05), whichwas different to that of seawater shrimps. At higher temperature, cyt-C contents infreshwater and seawater were higher than before (P<0.05).(2) Following temperaturedecrease, hemolymph caspase-3activity in freshwater shrimps increased significantly(P<0.05), while no significant change occurred in seawater shrimps was not observed(P>0.05). Caspase-3activity in hepatopancreas in freshwater shrimps changedsignificantly in lower temperature (P<0.05) and fluctuated irregularly in highertemperature. There was no significant change in the caspase-3activity inhepatopancreas in seawater shrimps (P>0.05).(3) cyt-C contents in hepatopancreas infreshwater and seawater shrimps increased significantly in low concentration ofnon-ionic ammonia, and increased first and then decreased in high concentration.(4)After the stress of low concentration of non-ionic ammonia, increased significantly(P<0.05). While in high concentration of non-ionic ammonia, for freshwater andseawater shrimps, caspase-3activity in hemolymph increased first and then decreased,but that in hepatopancreas kept increasing. In summary, the capability of adapting tolow temperature is higher in seawater shrimps than that in freshwater shrimps. Thephysiological response of freshwater shrimps to non-ionic ammonia stress was similarto that of seawater shrimps.