| Because of the influence of global warming and human activities,the natural environment has changed,and the phenotypic plasticity of amphibians is easily affected by the environment as well as regulated by genotype.Therefore,this study raises the following scientific questions: Under the changing environment,whether the phenotypic characteristics of amphibians will change and what will happen? If it has an impact on the entire population? How do genes regulate their phenotypic plasticity?To solve the above problems,this paper selected the environmental factors(temperature,steroid hormones)that have great effects on amphibians phenotypic to experiment,to further understand the effects of environment on amphibians phenotypic plasticity.At the same time,sex phenotype as the intuitive phenotype was selected to perform the transcriptome analysis,to explore amphibians phenotypic plasticity mechanism at the molecular level.Hoplobatrachus rugulosus is widely distributed in China and has important conservation and economic values.Therefore,this study mde relevant experiments with H.rugulosus as the experimental object,and obtained the following results:The experiment of temperature factor found that:(1)Too low temperature will affect the metamorphosis and survival rate of H.rugulosus tadpoles,which is not beneficial to the growth and development of H.rugulosus tadpoles.However,more larger individuals can be produced at low temperature,and 24? is already a low temperature environment H.rugulosus.(2)The higher the temperature,the smaller the individuals and the shorter the metamorphism time,but the tadpoles developed at 29?had more advantages in growth and reproduction performance.(3)High temperature can affect the gonad development of H.rugulosus,resulting in a male-biased sex ratio.The size of male and female H.rugulosus individuals is dimorphic,and female individuals are always larger than male individuals.(4)The development temperature of H.rugulosus has no significant effect on its offspring when it is mild or high,but the low temperature can affect the reproduction of H.rugulosus,making it unable to successfully breed offspring in the breeding period.The two-factor experiment of temperature × aromatase inhibitors(letrozole)was showed that:(1)34 ? could accelerate the growth and development of frog tadpoles and shorten the metamorphosis time,while the tadpoles reared at 29? grew slowly,but the metamorphosis larvae had larger individuals.The interaction of letrozole concentration,temperature and letrozole had no effect on these variables.(2)There was a positive correlation between the jumping distance and the condition factor of the juvenile frogs after metamorphosis,and the jumping distance was not affected by the temperature,letrozole concentration and their interaction when the condition factor was controlled.(3)The sex ratio of the control group(0 mg/g letrozole)was 1:1 at 29?,and more males were found at 34?.In the letrozole treatment group,the ratio of males was significantly biased at 29? and 34?,and the ratio of males increased with the increase of letrozole concentration.In addition,34? also produced more males per letrozole concentration than 29?.The transcriptome sequencing analysis of the gonads of H.rugulosus showed that:(1)There were significant differences in gene expression among different gonads.More genes are upregulated during development in the testicles than in the ovaries.(2)Compared with the immature female ovary,more genes were expressed in the mature ovary.More genes are upregulated early in testicular development.(3)Steroid hormones play an important role in gonadal development in H.rugulosus.Based on GO and KEGG differential gene enrichment analysis,this study identified the main regulatory pathway of steroid in sex development,cyp17?,hsd3?,hsd11?1,cyp19? and hsd17?12 are involved in gonadal development in H.rugulosus by regulating steroid hormones.(4)cyp3?,cyp17?,hsd3?,hsd11?1,sox2,sox9,sox30,soat,cyp19?,hsd17?12 and hsp?1s are all related to gonad development and differentiation.In conclusion,the growth and development of individual H.rugulosus is greatly affected by temperature,while the sex development of H.rugulosus is greatly affected by environmental temperature and steroid hormones,indicating that the global warming and the further aggravation of environmental pollution will have a great impact on the population of H.rugulosus,and may even cause the extinction of the species.Therefore,it is urgent to pay more attention to global warming and to prevent and control environmental pollution.In addition,it is also confirmed the important role of steroid hormones in the gonad development at the molecular level.Based on the results,further studies of the obtained genes can be conducted to explore the more specific molecular mechanisms in the process of sex development and gonad differentiation,and to refine the gene roles,laying a foundation for further studies in this field. |
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