Investigation On The Causes Of Death Of Crassostrea Hongkongensis In Spring And Identification Of Gonadal MicroRNAs

The large-scale death of Crassostrea hongkongensis in spring coincided with the maturity stage of the gonadal development.Salinity stress leading to accelerate gonadal development or obstacle gamete to discharge may be one of the death triggers.Mi RNAs have been found extensively in the regulation of gonadal development.But there are few studies on the gonadal mi RNAs of mollusks,especially in oysters.In this study,we investigated the cause of the mass death of C.hongkongensis in the oysters farming area of Guangxi province,and analyzed the relationship between death in spring and gonadal development.We constructed a mi RNAs library of gonad tissue of C.hongkongensis.The high-throughput sequencing was used to identify and analyze differentially expressed mi RNAs in gonadal tissue.Then we verified them with real-time quantitative PCR(RT-q PCR)method.These work established a foundation for the molecular regulation mechanism of gonadal development.The main findings are as follows:(1)The salinity in the area where large-scale deaths of C.hongkongensis occurred in spring sustained to be high.When the salinity of seawater is higher than 25‰,lasting for 16 days,C.hongkongensis died on a large scale.The mortality rate of C.hongkongensis was different at different sampling points in the same sea area.The higher the salinity was,the higher the mortality rate was.The gonads of C.hongkongensis in the diseased sea area matured compared to the low-salinity sea areas during the same period.According to the results,we speculated that the salinity stress may cause the gonads of C.hongkongensis maintaining at the stress state of maturation for a long time,then sperm and eggs cannot be effectively discharged by stimulating of freshwater,which would be the main reason for the large-scale death in spring of C.hongkongensis.(2)By high-throughput sequencing the ovarian and testis tissue of C.hongkongensis,we obtained yielded 13,634,997(ovarian)and 14,494,149(testicular)high-quality sequences about 18-35 nt by screening from s RNA library.We mapped these high-quality sequences to the C.hongkongensis transcriptome reference sequence,and identified 8,771,717(ovarian)and9,926,014(testicular)sequences corresponding to mi RNAs in the Rfam database.After blasting the mi RNAs sequences against the mi RBase database,we identified 50 known mi RNAs and 53 novel mi RNAs.(3)We analyzed the differential expression of 103 mi RNAs.Of these,27 mi RNAs were significantly upregulated in ovaries as compared to the testes,and43 mi RNAs were significantly upregulated in the testes as compared to the ovaries.Gene Ontology(GO)annotations indicated that target genes of the differentially expressed mi RNAs enriched in GO category such as the multi-organism cellular process,organelle part and protein binding.Additionally,the target genes were enriched in several KEGG pathways,including the ECM-receptor interaction,galactose metabolism,phagosome,and notch signaling pathway.It has been reported that these pathways play an important role in differentiation and maintenance of gonad.(4)To validate the differential expression results generated by high-throughput sequencing,we used RT-real-time quantitative PCR(RT-q PCR)to characterize the expression patterns of the six most differently expressed mi RNAs(lgi-mi R-1990,lgi-mi R-1986,lgi-mi R-263 b,lgi-mi R-279,lgi-mi R-1992,and novel-98)as well as two mi RNAs associated with gonad development(lgi-mi R-29 and lgi-mi R-8).Most of the RT-q PCR mi RNAs expression patterns were similar to those recovered by high-throughput sequencing with the exceptions of novel-98 and lgi-mi R-1992.It is indicated that sequencing analysis data is reliable and can be used for subsequent research.The analysis and identification of differentially expressed mi RNAs in the gonads of C.hongkongensis will help us understand the molecular regulatory mechanism of mi RNAs in the gonads development of C.hongkongensis.And we will develop the techniques for suppressing gonadal development based on the principles of molecular biology,as well as cultivate sterile series of C.hongkongensis to avoid the death in spring that may result from reproductive development.