| It has become a consensus to rejuvenate endangered wildlife resources through proliferation and release and has achieved remarkable results in the fields of fish and birds.As an amphibian,the Chinese giant salamander(Andrias davidianus)has been multiplied and released for several times in historical distribution areas,but due to the lack of scientific guidance,the effect is not obvious.In order to acquire the law of field activities,wild survival and growth status of released Chinese giant salamanders in suitable areas,and to find out the main restrictive factors that limit the proliferation and reintroduction,this study selected two sites(the Donghe River in the Ningshan County and the Heihe River located in Zhouzhi County)located at southern and northern slope of the Qinling Mountains to release the Chinese giant salamanders.At the same time,the real-time tracking and monitoring research of Chinese giant salamanders after proliferation and release was carried out with the help of radio telemetry.Meanwhile,a reasonable sex ratio is the guarantee of the successful multiply and release of Chinese giant salamander.However,due to late sexual maturity and unobvious gender characteristics during non-reproductive season,the release of Chinese giant salamander has a great blindness.Therefore,based on the sex-specific expression genes and the composition characteristics of sex hormone in urine,the research on the method of rapid gender identification of Chinese giant salamander that restrict the effect of proliferation was conducted,aiming to break through the problem of improper sex ratio caused by difficulty in gender identification of post-released Chinese giant salamander and provide assistance for multiply and post-release of Chinese giant salamander.The main research results are as follows:1.After 14 months of real-time wireless telemetry and tracking research,a comparative analysis of the survival rate of released Chinese giant salamander in the Heihe and Donghe fields showed that the survival rate of released Chinese giant salamander in the Heihe field(40.5%)was lower than that of the Donghe field(70.2%).The analysis of factors affecting survival rate showed that DSR(days held from surgery to release)and sites were the main factors affecting the survival rate of released Chinese giant salamander in the wild(ΔAICc < 2).The results of field growth monitoring indicated that the body length and weight of the released Chinese giant salamander at the two release sites were lighter than those of the artificial breeding individuals of the same population,but the difference was not obvious;the health check found that there were no parasites and viral diseases before and after release.Only in the early stage of release,some individuals were found to have local scratches,but slowly healed on their own in the later stage.The survey results of the bait abundance at the two release sites showed that the bait abundance of Heihe River field was significantly larger than that of Donghe River(p < 0.001).2.The study on the temporal and spatial distribution of released Chinese giant salamander in the wild environment had obtained 5,939 post-release site information,and the data analysis results showed that 4,441 location information comes from the Donghe release site.Chinese giant salamander released in the Donghe River had seasonal differences in the stationary rate,average daily moving distance,and home range.Compared with summer,in the winter,the stationary rate was higher,the moving distance was shorter,and the home range was smaller;the monitoring indexes in spring and autumn were between winter’s and summer’s,but there was no significant difference.The average dispersal distance of the Chinese giant salamander released in the Donghe River was 145.3 ± 61.9 m(n = 8),whereas the dispersal distances of the two surviving giant salamanders in the Heihe River were 211 m and 205 m.All individuals moving downstream were related to the impact of flood.In addition,based on movement ecology,mixed models were used to assess the biotic and abiotic variables that may affect the movement of Chinese giant salamander,including weight,body condition,temperature,precipitation and lunar phase.Model results showed that lunar phase,water temperature,and precipitation all had an impact on movement of the Chinese giant salamander,whereas body weight and physical condition had no significant impacts.This study revealed the environmental factors that affect the movement of released Chinese giant salamander,and also provided better suggestions for the release and conservation of Chinese giant salamander in mountainous areas.3.The gonads of Chinese giant salamander of different genders were sequenced using RNA-seq technology,15,063 differentially expressed genes(DEGs)were selected from the testes or ovaries,of which 7,973(52.93%)were highly expressed in the testis and 7,090(47.07%)in the ovary.A total of 20 DEGs were selected for q PCR verification,and the trend of gene expression was basically consistent with that of RNA-seq.According to the abundance of gene expression,13 DEGs were selected for expression detection in the muscle and skin tissues of the Chinese giant salamander of different genders.And 13 DEGs were detected in muscle and skin,and some genes especially Beta-microseminoprotein(Msp)presented a significant difference in the muscle of testis and ovary(p < 0.05),suggesting that detecting the expression of Msp in the muscle may become a potential biomarker of sex identification for Chinese giant salamander.4.In addition,through enzyme-linked immunoassay(EIA)analysis of the Estrone-3-Glucuronide(E1G)and Testosterone(T)of urine collected from Chinese giant salamander,the data demonstrated that the T average content of urine was significantly higher than E1 G in all male Chinese giant salamander(p < 0.05),and T average content of urine was significantly lower than E1 G in all female Chinese giant salamander(p <0.05),and the T/E1 G in the urine of adult male Chinese giant salamander was more than1.0;the ratio of the two hormones in the urine of adult female Chinese giant salamander was less than 1.0.The accuracy of gender identification of sub-adult Chinese giant salamanders was 83.33%.The results of T/E1 G ratio provided new insight for the future development of gender identification of Chinese giant salamander,and also laid the foundation for accurate sex identification of Chinese giant salamander that prepared for release after proliferation.In conclusion,firstly,wireless remote sensing for real-time tracking monitoring of artificial proliferation and release of Chinese giant salamander was carried out,the survival rate and growth status,spatial and temporal distribution,migration ability and activity sphere in home range of giant salamander in the field were mastered,and the factors affecting its movement in the wild was analyzed.Secondly,in response to the problem of gender identification restrictions in proliferation and release,non-damaging or minimal damage sampling was used for sampling,transcriptome sequencing and EIA technology were used for detecting of DEGs or sex hormone of gonads of different gender Chinese giant salamander.It was clear that it is possible to accurately identify Chinese giant salamander gender through specifically expressed genes DEGs or sex hormones,which opens up a new direction for our next research on the sex of Chinese giant salamander. |
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