| Examination of physiological responses of mussels to thermal stress is crucial to evaluate their biogeographic distribution and adaptation ability in the changing climate.In the present study,we investigated the impacts of cold and heat stress on physiological responses of two mussel species,blue mussel Mytilus galloprovincialis and green mussel Perna viridis.Firstly,the effects of temperature stress(M.galloprovincialis:6℃,10℃,18℃,22℃,14℃ as the control;P.viridis:18℃,22℃,30℃,34 ℃,26 ℃ as the control)on the early development of mussels were studied.Our results showed that mussels’ embryos could not hatch after cold stress,and a decrease in hatching rate and higher malformation rates of embryos and larvae of mussels were occurred after heat stress.A significantly decreased survival rate of D-shaped larvae of two species mussels was showed after acute cold and heat stress.Reactive oxygen species(ROS)and malondialdehyde(MDA)content increased significantly in the early development stages of mussels under cold and heat stress conditions(p<0.05),and the capacity of antioxidant was also enhanced as the development progressed.The transcriptions of β-catenin and nacrein were signifcantly up-regulated in embryos of mussels after cold and heat stress(p<0.05).Furthermore,the transcripts of hsp70 and grp78 were significantly increased in the early development stages of mussels after heat stress(p<0.05).A significant down-regulation of parp expression in mussels’ fertilized eggs was detected after cold and heat stress(p<0.05),and DNA damage was also occurred.P450 transcripts in embryos of mussels were significantly increased after cold and heat stress(p<0.05).The early stage of the development of M.galloprovincialis is more actively in response to temperature stress,and the ability of P.viridis to repair DNA damage may be stronger.Cold and heat stress(M.galloprovincialis:4℃,8℃,25℃,35℃,15℃ as the control;P.viridis:8℃,15℃,35℃,42 ℃.25 ℃ as the control)induced a temperature-dependent increase of the mortality rate in two mussel species.Oxygen consumption rate and ammonia excretion rate of mussels increased with increasing of temperature,but declined when the temperature exceeded the mussels’ tolerance threshold.Gill structure in mussels damaged by acute temperature stress,and this damage is irreversible.THCs and cell viability decreased significantly in M.galloprovincialis after 35℃ stress(p<0.05),and it occurred in P.viridis at 8℃ stress.It is suggested that M.galloprovincialis may respond to heat stress by reducing oxygen consumption and accelerating catabolism,while P.viridis increase oxygen consumption and reduce catabolism to cope with it.ROS production in the gill tissue and hemocytes of M.galloprovincialis increased significantly after cold stress at 4℃,and ROS in hemocytes increased at 35 ℃ stress.However,ROS increased significantly in the gills and hemocytes of P.viridis after cold and heat stress.MDA content and mitochondrial membrane potential(MMP)were increased significantly in the gill tissue and hemocytes of both species after cold and heat exposure(p<0.05).Single-stranded DNA damage was more serious in the hemocytes of two species mussels after cold stress,and a higher damage of double-stranded DNA was induced by heat stress.The results suggest that oxidative damage in M.galloprovincialis after temperature stress may be lighter than that of P.viridis.Extreme cold stress may cause more serious damage to the hemocytes of M.galloprovincialis,while P.viridis intensifies after heat stress.The activities of antioxidant enzymes(SOD,CAT and GSH-Px)increased significantly(p<0.05)in the gill tissue of M.galloprovincialis,but strongly up-regulated in P.viridis after heat stress.The transcripts of hsp70 up-regulated in the gills of mussels under heat stress.The phosphorylation level of p38-MAPK was increased significantly(p<0.05)in the gill tissue of P.viridis after cold and heat stress,but only increased in M.galloprovincialis after cold stress.However,the activation of caspase-3 was detected in the gills of the two species after extreme cold stress.The anti-oxidative reaction of M.galloprovincialis may be stronger than that of P.viridis,and the difference in the p38-MAPK phosphorylation levels between two species of mussels may be one of the main factors influencing the temperature tolerance.Cold and heat stress also indcued antioxidant enzyme activities increased in hemocytes.hsp70 transcripts of M.galloprovincialis in hemocytes were up-regulated under 25℃ stress,but increased in P.viridis after cold and heat stress.Additionally,TUNEL assay found that the apoptosis of hemocytes was increased in M.galloprovincialis after cold stress,while the apoptosis rate of P.viridis was higher under heat stress.Two bcl-2 cDNA sequences were identified and characterized from P.viridis,which named as Pvbcl-2A and Pvbcl-2B.The full-length cDNA of Pvbcl-2A contained a 558 bp open reading frame(ORF)encoding for 186 amino acids with a calculated molecular weight(Mw)of 45.74 kDa and pI of 4.47.Pvbcl-2B included an ORF of 555 bp encoding a polypeptide of 185 amino acids with a calculated Mw of 45.3 kDa and pI of 5.29.Both Pvbcl-2A and Pvbcl-2B contained four highly conserved bcl-2 homology(BH)domains.Sequence and phylogenetic analysis demonstrated that Pvbcl-2A and Pvbcl-2B were clustered in the same clade with a closest relationship to other mollusks bcl-2 members.After temperature stress,significantly increased bcl-2 transcripts of two species mussels were detected in the gill tissue(p<0.05).Mgcyt-c showed transcriptional down-regulation in the gills after cold and heat stress(p<0.01),but increased Pvcyt-c transcripts were detected.Mgbcl-2 transcripts in hemocytes showed significantly increased after cold and heat stress(p<0.05).However,Pvbcl-2A and Pvbcl-2B showed transcriptional down-regulation in hemocytes after acute cold and heat stress(p<0.01),and Pvcyt-c transcripts up-regulated after heat stress.After bcl-2 inhibitor ABT-263 treatment,both Pvbcl-2A and Pvbcl-2B transcripts in gill tissue decreased significantly after cold exposure at 15℃(p<0.05).However,the increased transcripts of both Pvbcl-2A and Pvbcl-2B were not being inhibited at 25℃ and 35℃ exposure.Nevertheless,phosphorylation levels of p38-MAPK increased significantly in the gills after ABT-263 treatment under both cold and heat exposure(p<0.05).No significant activation of caspase-3 was detected after inhibitor treatment.The full-length cDNAs of Pvbax and Pvbak were 1020 bp and 1131 bp,and respective encoding for 210 and 234 amino acids with a conserved BCL domain and transmembrane region.Sequence and phylogenetic analysis demonstrated that Pvbax and Pvbak were clustered in the same clade with a closest relationship to other mollusks members.A significant decreased in the gill tissue of Mgbax and Mgbak transcripts were occurred after cold stress(p<0.05),but they increased significantly after heat stress(p<0.05).The transcriptions of Pvbax and Pvbak were significantly down-regulated in the gill tissue after cold stress(p<0.05),but they significantly up-regulated after heat stress(p<0.05).The transcripts of Mgbax and Mgbak in hemocytes were significantly up-regulated after cold and heat stress during recovery period(p<0.05).The expression of Mgbax in the hemocytes was significantly decreased after heat stress;however,the expression of Mgbak was significantly increased(p<0.05).The transcription of Pvbax in the hemocytes significantly increased after heat stress(p<0.05),but the transcription of Pvbak was significantly down-regulated(p<0.05).It is shown that the response of bax and bak in the hemocytes of two species mussels to temperature stress was different,and cold stress may induce apoptosis of hemocytes in M.galloprovincialis more easily.Temperature stress causes inadequate reprogramming early development of mussels.The responses of embryos in mussels to temperature stress are more positively,so that the adaptability to stress and the ability to repair cell damage may be improved.Antioxidant enzymes,hsp70,p38-MAPK and apoptosis-related factors were involved in the regulation of mussels after temperature stress.The oxidative damage of M.galloprovincialis was lower than that of P.viridis,and its physiological responses to cold stress were stronger.Therefore,M.galloprovincialis may be more resistant to temperature stress than P.viridis,which explains the reason why M.galloprovincialis can be moved southward gradually. |
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