Study Of Physiological Response And Differential Gene Expression In Pomacea Canaliculata(Mollusca:Gastropoda) Under Low Temperature Stress

The golden apple snail,Pomacea canaliculata(Lamarck,1822),is an invasive freshwater snail originating from South America.In China,the P.canaliculata was found to North latitude 31.0 N and is widely distributed across more than 15 provinces.Low temperature in winter is the direct obstacle for the disperse and survive of this snail.The cold tolerance of snail is one of the key factors that restrict the occurrence,distribution and diffusion of snail populations.The adaptability of P.canaliculata to low temperature has been well investigated,however,very little research was focused on the molecular mechanisms of P.canaliculata under cold stress.In this paper,we study the physiological response and biochemical changes of overwintering P.canaliculata,and their relative gene expression and cold-tolerance.The results are as follows:1.The results showed that the SCP(supercooling points)of overwintering juvenile snails was significantly positively correlated with the ambient temperature.The changes of SCP were closely related with the temperatures at the sampling locations.The SCP tended to decrease initially as the environmental temperature decreased,and reached the lowest value on February.The SCP of this snail ranged from-7.14 C to-6.74oC,with a mean of-7.29oC.The cold tolerance of the overwintering juvenile snails increased with the decrease of the environmental temperature,indicating the cold tolerance can be improved by cold acclimation.The SCP and Freezing point of juvenile snails were affected by their body size,simply it could be monitored by shell size.The SCP of juvenile snails with a shell height of 12.6 mm≤H<20mm was significantly lower than those with heights of 5 mm≤H<10.5 mm,10.6 mm≤H<12.5 mm and 23 mm≤H<45 mm,respectively,suggesting that the juvenile snails with the height of 12.6 mm≤H<20mm had a higher cold tolerance.2.The antioxidant enzyme activities of juvenile snails of P.canaliculata under low temperature were studied.The results showed that the antioxidant enzyme related to stress resistance,including superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPX),played an important role in protecting the body against the injury from cold.With the extension of cold stress,the activities of three enzymes(SOD,CAT and GPX)increased gradually,reached the maximum value at 12 h,and then decreased gradually.The content of MDA gradually increased and reached the maximum value at 24 h,then decreased gradually,but it was still higher than the control(P<0.05).The activity of Na+//-K+-ATPase decreased significantly during the first 24 h and then increased gradually till day 5,and remained decreased significantly to the control(p<0.05).The regularity of the activities of SOD,CAT,GPX as well as MDA and Na+/-K+-ATPase under low temperature indicated that the above mentioned enzymes were involved in response to the low temperature.3.The physiological indices and the enzyme activities involved in the overwintering stage were studied in juvenile snails of P.canaliculata in each month during the winter.The determination of physiological indices and enzyme activities showed:l)Glycerol and glucose were considered as the potential cryoprotectants,which showed a significantly negative correlation with the SCP during the overwintering.2)Glycogen was considered as the important energy substances,which decreased obviously in mid-overwintering period.3)The total water content of juvenile snails tended to decrease gradually with the decrease of environmental temperature,and reached the lowest on February.Subsequently,with the rising of seasonal temperature of late overwintering,the juvenile snails were released from dormancy and the physiological metabolism is gradually enhanced,the water content was gradually increased.4)The content of lipid increased gradually,reached the maximum value on November,and then decreased gradually.5)There were seasonal differences in the activities of metabolic enzymes(pyruvate kinase PK,lactate dehydrogenase LDH,glycerol kinase GK and Glycerol-3-phosphate dehydrogenase,GPDH)during the overwintering.The activity of PK increased significantly in the overwintering stages and reached the maximum value on February,then decreased gradually.The activity of LDH gradually decreased during the overwintering and reached its minimum value on February of next year,and then increased with the increase of environmental temperature,indicating that their cold tolerance decreased.GK and GPDH are two key enzymes in the glycerol metabolism pathway.The activity of GK decreased gradually from September to November,increased gradually in December and reached the maximum on February of next year.The activity of GPDH increased during the wintering,reached the maximum,on February of next year,and then decreased.These seasonal differences suggest that the above mentioned metabolic enzymes may play an important role in regulating the cold tolerance of juvenile snails of P.canaliculata.4.A SSH library of juvenile snails under low temperature(0℃)and normal temperature(25℃)was constructed.After dot blot hybridization analysis and sequencing,we obtained 107 and 95 non-redundant differentially expressed sequence tags(ESTs)from the forward and reverse libraries,respectively.BIast2GO analyses indicated that 95 ESTs,35 from the forward library and 60 from the reverse library,displayed a significant sequence homology to genes with known or putative functions.They were categorized into seven physiological functions including stress resistance,signal transduction,posttranslational modification/chaperones,carbohydrate transport and metabolism,energy production and conversion,enzyme regulator activity and translation,ribosomal structure.5.RT-PCR and quantitative real-time PCR were used to detect the transcript levels of HSP70,GK,GPDH and NKA in juvenile snails under low temperature with different time treatments,and the HSP70,GK,GPDH and NKA genes were knocked down by RNAi to study the influence on mortality of snails.The results showed that the expression level of hsp70 was significantly up-regulated under cold stress until day 3,and then exhibited a slight decline at day 5.The transcript of Na+/K+ATPase was down-regulated significantly during the first 24 h and then increased gradually till day 5,and remained decreased significantly compared to the control.Expression patterns of GPDH and GK were largely similar,although the time frames varied.Both RT-PCR and qRT-PCR results of all candidate genes showed a consistent with SSH results.The expression level of dsRNAi-HSP70,dsRNAi-GK,dsRNAi-GPDH and dsRNAi-NKA decreased significantly,while the transcript levels of four genes in the non-injected remained unchanged.After low temperature exposure,the mortality of the dsRNA-treated juvenile snails was remarkably higher than that of the non-injected controls.