Physiological Responses To Temperature And Salinity Stress In The Sea Cucumber Apostichopus Japonicus And Intertidal Marine Snails (Chlorostomas And Littorina)

A series of experiments were conducted to investigate the effects of temperature andsalinity stress on physiology, growth, survival and distribution of the sea cucumberApostichopus japonicus and intertidal marine snails (Chlorostomas and Littorina). Theprimary results were listed below.1Effects of thermal and osmotic stress on the growth in the sea cucumberApostichopus japonicus, and its physiological mechanismsThis study deals with the effects of thermal and osmotic stress on growth,osmoregulation and Hsp70level in the sea cucumber Apostichopus japonicus Selenka.Sea cucumbers were exposed to30C (Hs treatment) or20C (control) for2h, and thenwere challenged with osmotic shock, which was administered by transferring the seacucumbers from natural seawater (about32ppt) to different salinities (20,25,30and40ppt). After a30-day experiment, the specific growth rate (SGR) and survival rate of seacucumbers were measured. To elucidate the underlying physiological mechanisms,osmotic pressure of the coelomic fluid, Na+, K+-ATPase and Hsp70level of the seacucumbers from Hs treatment and control were measured at selected times (1,2,6,24,and48h) after the osmotic shock. At the end of the30-day experiment, SGR of Hs seacucumber was higher than that of the control at the salinity of20ppt. However, SGR ofHs sea cucumber was lower than that of the control at the salinity of30,40ppt and naturalseawater. At the salinity of20ppt, the survival rate of A. japonicus was only20%. These results indicted that a salinity of20ppt exceeded the ecological tolerance for growth injuvenile A. jaoponicus. A prior heat shock could accelerate growth when the ambientsalinity was below the ecological tolerance for growth (salinity of20ppt); on the otherhand, it could retard growth when the ambient salinity was within the ecological tolerancefor growth (salinity of25,30,40ppt and natural seawater). The osmotic pressure of thecoelomic fluid changed rapidly and stabilized by6h after the osmotic shock. Thetemporal change pattern of Na+, K+-ATPase activity increased initially and reached themaximum value within6h. These results indicated that A. japonicus was an osmoticconformer, and the activity of Na+, K+-ATPase had an adaptive response to the change ofambient salinity. Both thermal and osmotic stresses could induce the up-regulation ofHsp70, which might partly explain the differences in growth of sea cucumbers at differentsalinities.2The effects of acute salinity decrease on the survival and hsp70expression injuvenile sea cucumber Apostichopus japonicusThe mortality of juveniles of the sea cucumber Apostichopus japonicus and relatedphysiological responses (osmoregulation and heat shock protein70(Hsp70) geneexpression) were studied in a simulated field salinity decrease after a heavy rainfall insummer. Salinity firstly decreased gradually from30ppt to20ppt (S20) or25ppt (S25) ata rate of2.5ppt every6h, and then was maintained at20ppt or25ppt. After96h at thelow salinities, salinity was increased gradually to30ppt at a rate of2.5ppt every6h, andthen was maintained at30ppt for96h. There was no mortality during salinity decreases,but40%~50%mortality occurred when salinities were maintained at low levels. Theosmotic pressure of the coelomic fluid changed with decrease in ambient salinity butstabilized within6h after the salinity change. In both S20and S25treatments, expressionof hsp70mRNA increased initially after salinity decrease, reached amaximum value at the lowest salinity and returned to the control level (without salinity decrease) after72-hlow-salinity exposure. These data suggest that low salinity can cause large-scale mortalityof juvenile A. japonicus, which may be partly due to the rapid decrease of osmoticpressure in the coelomic fluid during hypo-osmotic stress. Furthermore, cellular levelstress is indicated by up-regulation of mRNA for the molecular chaperone Hsp70, but thisadaptive response can only provide temporary protection against salinity decrease.3Viserosomatic index and RNA/DNA ratio in aestivating sea cucumber ApostichopusjaponicusThe viserosomatic index and RNA/DNA ratio in the sea cucumber Apostichopusjaponicus under aestivation were investigated. For the experiment group, the watertemperature was increased gradually from16℃to26℃within two weeks, andmaintained at26℃for five weeks. For control group, the temperature was kept at16℃for seven weeks. During the experiment period, five individuals from each group weresampled every week, and the viserosomatic index and RNA/DNA ratio were measured.The results showed that the viserosomatic index in the sea cucumbers from experimentgroup decreased gradually as the temperature increased, became significantly lower thanthat from control group after one-week aestivation, and then decreased gradually to arelatively low level. The RNA/DNA ratio of the three tissues (body wall, intestine andrespiratory tree) in the sea cucumbers from the experiment group shared the same trend.The RNA/DNA ratio increased with the temperature, became significantly higher than thatfrom control group after one-week aestivation, and then decreased gradually. The increaseof RNA/DNA ratio was probably related to the up-regulation of proteins which wereinvolved in heat resistance. 4The effects of acute salinity decrease on the survival and hsp70expression in adultsea cucumber Apostichopus japonicus under aestivationThis study deals with the mortality and related physiological responses of aestivatingsea cucumber Apostichopus japonicus to acute salinity decrease. Aestivating and activesea cucumbers were exposed to a decrease in salinity (from30ppt to20ppt) at a rate of2.5ppt every6h, and then maintained at20ppt for96h. The mortality of aestivating seacucumbers was~30%, which was significantly higher than that of active sea cucumbers(~10%). This result indicated that sea cucumbers in aestivation were more susceptible tohypo-salinity stress. To elucidate the underlying physiological mechanisms, the osmoticpressure in coelomic fluid and the levels of hsp70and hsp90mRNA in aestivating andactive sea cucumbers were measured. No significant difference in osmoregulation wasobserved between the two groups. The osmotic pressure of coelomic fluid in both groupschanged with decrease in ambient salinity and stabilized at the levels similar to ambientenvironment within6h after the salinity decrease. There were significant differences inthe time course and magnitude of hsp70and hsp90expression between the two groups.After exposure to decreased salinity, aestivating sea cucumbers showed a delayedup-regulation of hsp70and hsp90expression compared to animals in active state, andthese levels decreased rapidly to control values. The expression of hsp70and hsp90inaestivating sea cucumbers were significantly lower than those in active sea cucumbersafter salinity change. The differences in hsp70and hsp90expression between the states may partly explain the higher mortality of sea cucumbers in aestivation when exposed tolow salinity.5The effects of acute temperature and salinity change on osmoregulation and heatshock proteins gene expression of the sea cucumber Apostichopus japonicus SelenkaThe effects of acute temperature and salinity change on the osmoregulation and heatshock protein gene expression in the sea cucumber Apostichopus japonicus wereinvestigated. The experiment was conducted at different temperature (16,20,24and28℃)and salinity (22,27and32ppt) combinations. The results showed that high temperaturecan accelerate the change of osmotic pressure in the coelomic fluid when salinitydecreased; at a same salinity, hsps expression increased with temperature increase; at asame temperature, hsps expression increased firstly and then decreased with salinitydecrease. Two-way ANOVA analysis showed that compared to salinity, the effect oftemperature on hsp expression was more prominent.6Temperature adaptation of cytosolic malate dehydrogenases for Chlorostomas andLittorina marine snails: thermal sensitivity of protein correlates with thebiogeographic distributionWe studied thermal adaptation of cytosolic malate dehydrogenase (cMDH) from themarine snails (Chlorostomas and Littorina) that have different intertidal verticaldistribution. As an index of adaptation of function, we measured effects of temperature onthe Michaelis-Menten constant of cofactor NADH (KmNADH). KmNADHvalues of cMDHfrom low-intertidal species C. brunnea and C. montereyi were more sensitive totemperature increase than the mid-intertidal species C. funebralis and C. rugosa. cMDHof L. keenae and L. scutulata are the least sensitive to temperature increase. Thermal stability (rate of loss of activity at42.5°C) showed a similar pattern of interspecificvariation. To investigate the underlying molecular mechanisms, we compared the aminoacid sequences of cMDH from these species, anlalzyed them with phylogenetic analysisby maximum likelihood (PAML) and constructed a three-dimensional model of cMDHmonomer based on the ternary complex of pig cMDH. The results indicated that multiplesites in the protein, notably those that may influence conformational mobility duringbinding, may be sites of adaptive change.