Effects Of Temperature On Embryonic Development And Immune Function Of Turtles

Turtles are ectotherm,the temperatures have important effects on embryonic development,incubation duration,hatching success,hatchling phenotypes,post-hatching growth,and immune function.The substrate moisture also has important effects on hatching success,incubation duration,and hatchling phenotypes during eggs of turtles incubated.In the turtle culture practice,it is found that the greenhouse-reared turtles are vulnerable to illness and death in temperatures change,whereas outdoor pond-reared turtles often display have well adaptation of temperatures change.The Mauremys reevesii and Mauremys mutica have been widely-cultured in China and widely used as experimental materials for scientific research such as ecological evolution due to their important scientific and economic value.At present,some studies have been carried out on the effects of temperature and moisture on the embryonic development in M.reevesii and M.mutica.However,the range of temperature setting in the existing studies is still relatively limited,especially the lack of studies under high and low temperature stress,and the lack of research on the interaction effects of temperature and moisture.In terms of immune function,there is no research on the effects of temperature stress on the differences of individual immune function between greenhouse-reared turtles and outdoor pond-reared turtles.The research of the effects of temperature on the embryonic development and immune function of turtles is helpful to reveal the law of temperature adaptation of turtles.The results have important reference value for the improvement of turtle culture conditions.Therefore,in this study,on the one hand,the effects of temperature and moisture on embryonic development and hatchling phenotypes of M.reevesii were studied by using precise temperature control technology.On the other hand,the effects of temperature on the immune-related enzyme activities of M.mutica,and the difference of immune response in the greenhouse-reared and outdoor pond-reared M.mutica under temperature stress were studied by using benzene disodium phosphate method,and Quantitative Real-time PCR technology.The main results include the following four parts:(1)The suitable temperature and moisture condition of embryonic development in M,reevesiiIn this study,eggs of M.reevesii are incubated at five temperature levels(23,26,29,32 and 35,range ± 0.5?).In each temperature treatment,there were three substrate moisture levels(1:0.5,1:0.9,and 1:1.2,weight ratio of vermiculite vs water).Thus,a total of 15 combinations of temperature and moisture are performed,aiming to examine the effects of incubation temperature and substrate moisture on embryonic development,hatchling phenotypes,locomotor performance,post-hatching growth,and hatchling survival.The results indicate that incubation duration decreased nonlinearly as temperatures increasd,the relationship between incubation duration(ID)and temperature(T)can be expressed by the following equation:ID=0.5498T2-36.137T+654.56.Eggs incubated at 23?,26? and 29? show higher(p<0.05)hatching success than those incubated at 32? and 35?.Deformity rate of hatchlings are significant higher(p<0.05)at 35? than the other temperatures.Hatchlings incubated at 23? and 26? were significant larger in body size and mass than those incubated at 29? and 32?.The locomotor performance is better incubated at 26? and 29? than that incubated other temperatures.However,the body mass and locomotor performance have no significant difference(p>0.05)among incubation temperature levels when the turtles are 3 months of age.At 12 months of age,hatchlings incubated at 23? and 35? have lower survival rates than those incubated at 26?,29? and 32?(p<0.05).For moisture effects,substrate moisture has significant effects on incubation duration,and eggs incubated in high(1:1.2)moisture level have shorter incubation duration than that incubated in low(1:0.5 or 1:0.9)moisture levels,while moisture have no significant effects(p>0.05)on hatching success,hatchling phenotypes,locomotor performance,post-hatching growth and hatchling survival.The results indicate that the combination of substrate temperature(29?)and moisture(1:1.2)represented optimal incubation conditions in this experiment.(2)The effects of low temperatures on embryonic diapause and embryonic development in M.reevesiiIn this study,the eggs of M.reevesii are incubated at five low temperature levels(13,15,17,19,and 21,range ± 0.5?)and ten treatment times(5 days,10 days,15 days,20 days,25 days,30 days,45 days,60 days,75 days,and 90 days),aiming to examine the effects of different low temperatures and treatment times on embryonic diapause and embryonic development.The results indicate that incubation duration of each treatment group significantly shorter(p<0.05)at 15?,17?,19? and 21? than the control group(30?),incubation duration decreased as treatment times increasd,indicating that the embryos are still developing slowly under these temperature conditions.Incubation duration of each treatment group is not significantly different(p>0.05)between 13? and the control group,indicating that the embryos are diapause at 13?.At 21?,hatching success is significantly shorter(p<0.05)at four time points(5 days,10 days,25 days and 30 days)than the control group,the deformity rate of hatchlings is significantly higher(p<0.05)than that the control group when the treatment time is more than 60 days.At 19?,hatching success is significantly shorter(p<0.05)at six time points(5 days,30 days,45 days,60 days,75 days and 90 days)than the control group,the deformity rate of hatchlings is significantly higher(p<0.05)than that the control group when the treatment time is more than 30 days.At 13?,15? and 17?,hatching success is significantly shorter(p<0.05)at nine time points(except 5 days)than the control group,at 15? and 17?,the deformity rate of hatchlings is significantly higher(p<0.05)than that the control group when the treatment time is more than 10 days,at 13?,the deformity rate of hatchlings is significantly higher(p<0.05)than that the control group when the treatment time is more than 5 days.The results indicate that hatching success gradually decreased and the deformity rate of hatchlings gradually increased as temperatures decreased and treatment times increased.(3)The effects of temperatures on the activity of two non-specific immune-related enzymes and survival rate in M.muticaIn this study,the survival rate of 1 and 2 age of M.mutica are monitored under three temperatures(24?,28? and 32?),and the activity of immune-related enzymes in serum is detected by sampling at 30 days,60 days,90 days,120 days and 150 days,respectively.We compared the differences in enzyme activity to reveal the effect of different temperatures on immune function of M.mutica.The results show:in 1 age of M.mutica,at 32?,the survival rate significantly lower(p<0.05)than at 24? and 28? after 90 days of rearing,and the activity of acid phosphatase(ACP)and alkaline phosphatase(AKP)in serum significantly lower(p<0.05)than at 24? and 28? at 120 days of rearing;in 2 age of M mutica,at 32?,the survival rate,ACP and AKP significantly lower(p<0.05)than at 24? and 28? at 150 days of rearing.The results indicate that high temperature(32?)inhibited the activity of the serum ACP and AKP of M.mutica,speculation that high temperature stress may inhibit the non-specific immune ability of the organism,this may be one reason for the increased death rate.(4)Response of transferrin gene expression to temperature stress in greenhouse-rearing and outdoor pond-rearing of M.muticaIn this study,greenhouse-reared turtles(GRTs)and outdoor pond-reared turtles(OPTs)are transferred into 24?(acute cold stress,ACS)and 32?(acute heat stress,AHS),respectively.Three turtles are sampled at each time point(3 h,6 h,12 h,24 h and 48 h after treatments),the individuals were not treated with ACS or AHS and were used as a control group(28?),the expression pattern of transferrin(TF)gene in liver was detected.The results show that for GRTs,the expression levels of an immune-related gene for transferrin were significantly different(p<0.05)between the control group and the ACS group for most time points(3 h,6 h and 48 h),while at two time points(6 h and 12 h)there was a significant difference(p<0.05)between the control group and the AHS group.However,for OPTs,we found the opposite pattern:the ACS group showed no significant difference(p>0.05)from the control group for all time points(3 h,6 h,12 h,24 h and 48 h),whereas two time points(12 h and 24 h)were significantly different(p<0.05)for the AHS group.Our results indicate that ACS may influence the immunity of GRTs and have no influence on OPTs,whereas AHS may largely affect the immunity of OPTs and have little influence on GRTs.