The Effect Of Water Temperature On The Growth And Gonadal Differentiation Of Giant Salamander Larvae And The Expression Analysis Of Wnt4 Gene

Amphibious animal gonadal differentiation is a hot research topic in developmental biology.Amphibians belong to thermotropic animals.Their growth and gonad differentiation are affected by environmental temperature.It is of great theoretical value for amphibian protection to study the effect of temperature on the growth and gonad differentiation of amphibians.In this paper,three water temperature gradients were set up within the suitable water temperature range for the growth of Andrias davidianus larvae.The body weight of the larvae of Andrias davidianus was measured three times under each water temperature gradient.The sex identification of the larvae was carried out by paraffin section and HE staining.The aim was to explore the effect of water temperature on the growth of larvae and gonad differentiation of Andrias davidianus.At the same time,according to the high-throughput transcriptome sequencing data in our laboratory,the differentially expressed genes were screened,and the gene Wnt4 related to gender differentiation was selected.The expression of Wnt4 in testis and ovary of giant salamander larvae was localized by in situ hybridization technology.The expression of Wnt4 was quantified by real-time quantitative PCR and Western Bolt technology,so as to explore the expression of Wnt4.Differences in the expression of Wnt4 in testis and ovary of giant salamander larvae and the effect of water temperature on the expression of Wnt4.The main results and conclusions are as follows:1.Effect of water temperature on larval growth of giant salamander.The larvae of giant salamander grew slowest at 14?,followed by 24? and fastest at 21?.The growth rate of larvae of giant salamander was significantly different under three water temperature gradients(P<0.05).Thc results showed that the growth rate of giant salamander larvae increased with the increase of water temperature in the water temperature range of 14?21 ?,but the growth of giant salamander larvae was inhibited when the water temperature was higher than 21?.2.The effect of water temperature on the percentage of gonadal differentiation of giant salamander larvae.The larvae of 9 giant salamanders collected at 14? were 3 ?6?with a sex ratio of 1:2 and a female rate of 33%.The larvae of 9 giant salamanders were 6?3?,the sex ratio of male to female was 2:1,and the female ratio of the two groups increased significantly to 67%at 21? and 24?.The results showed that high water temperature could make the gonad differentiation of giant salamander larvae tend to females,while low water temperature could make the gonad differentiation of giant salamander larvae tend to males.3.The effect of water temperature on the differentiation time of gonad in giant salamander larvae.Under three water temperature gradients,the differentiation time of the gonads of larvae of giant salamanders with membrane outlet for about 327 days was quite different:?Some of the gonads of larvae of giant salamanders in 14? water temperature group initially differentiated into primitive follicles,and the gonads of individual larvae of giant salamanders had not yet differentiated.?The gonads of larvae of giant salamanders in water temperature groups of 21? and 240C have fully differentiated and the degree of differentiation is obvious.It is suggested that the gonad differentiation of giant salamander larvae is sensitive to the change of water temperature.High water temperature can advance the gonad differentiation time and accelerate the process of gonadal differentiation.4.Location of Wnt4 in the testis and ovary of giant salamander larvae.The strong positive expression of Wnt4 mRNA in ovaries of 14 C group was mainly concentrated in somatic cells at the margin of ovary and tubular epithelial cells connected with ovary.The expression of Wnt4 was weak in testis,mainly in tubular cells at the junction of renal medulla and cortex connected with testis.The strong positive expression of Wnt4 mRNA in ovaries of 240C group was mainly concentrated in follicular fineness.The positive expression of Wnt4 in testis was mainly concentrated in spermatogonia and somatic cells near the immature zone of the middle kidney at the base of the testis,spermatocgonia in the seminiferous lobule and somatic cells in the connective tissue in the middle of the cross section of the testis.5.Analysis of Wnt4 mRNA expression in testis and ovary of giant salamander larvae.Real-time quantitative PCR was used to detect the expression of gene from high to low in the order of 140C female salamander>21 Cmale salamander>21 ? male salamander>24 ? male salamander>14? male salamander.The expression of Wnt4 gene in 140Cgroup was significantly different between male and female salamanders(P<0.05),and the expression of Wnt4 gene in female salamanders was about twice as high as that in male salamanders.The Wnt4 expression level was not significantly different between male and female salamanders(P>0.05)at 21 C and 24 C.Wnt4 expression was highest in female salamanders at 14 C and lowest in male salamanders at 14 C.At the same water temperature,the expression of Wnt4 mRNA in female salamander larvae was always higher than that in male salamanders.Therefore,we believe that the expression of Wnt4 is closely related to the development and functional maintenance of larval ovaries of giant salamanders.6.Analysis of Wnt4 protein expression in testis and ovary of giant salamander larvae.Western Bolt results showed that the order of Wnt4 protein expression from high to low was:Analysis of Wnt4 protein expression in testis and ovary of giant salamander larvae.Western Bolt results showed that the order of Wnt4 protein expression from high tolow was:14 Cfemale salamander>14? male salamander>21? male salamander>24 C female salamander>21 ? female salamander>24? male salamander.At the same water temperature,the expression of Wnt4 protein between female and male salamander larvae of two groups was significantly different(P<0.01),and the expression of Wnt4 protein between male and female salamander was significantly different(P<0.05).At the same water temperature,the expression of Wnt4 protein in female salamanders was higher than that in male salamanders at 140C and 24? degrees centigrade,but the expression of Wnt4 protein in male salamanders at 21 ?degrees centigrade was higher than that in female salamanders,and the diferencr was very significant(P<0.01).Therefore,the expression of Wnt4 is not gender-specific.Wnt4 not only participates in the development of ovary,but also is a part of the testis development pathway of larvae of giant salamander.14? female salamander>14? male salamander>21? male salamander>21? female salamander>24? female salamander>24? male salamander.At the same water temperature,the expression of Wnt4 protein between female and male salamander larvae of two groups was significantly different(P<0.01),and the expression of Wnt4 protein between male and female salamander was significantly different(P<0.05).At the same water temperature,the expression of Wnt4 protein in female salamanders was higher than that in male salamanders at 14 C and 24? degrees centigrade,but the expression of Wnt4 protein in male salamanders at 21 ? degrees centigrade was higher than that in female salamanders,and the difference was very significant(P<0.01).Therefore,the expression of Wnt4 is not gender-specific.Wnt4 not only participates in the development of ovary,but also is a part of the testis development pathway of larvae of giant salamander.7.Effects of water temperature on Wnt4 expression.This study showed that the gonadal differentiation of giant salamander larvae was immature at 14?,and the expression of Wnt4 mRNA and Wnt4 protein in ovary was the highest at this temperature.With the increase of water temperature,the differentiation of gonad of giant salamander larvae is more mature,but the expression of Wnt4 mRNA and Wnt4 protein decreases.Therefore,we speculate that Wnt4 plays a more important role in the early stage of ovarian development of giant salamander larvae.