Histological Observation And Transcriptome Analysis Of The Three Mantle Folds In Mytilus Coruscus

Mytilus coruscus are one of the important species in China’s artificial breeding industry.Its advantages of fast growth,strong disease resistance and low breeding cost make it highly recognized by farmers.As an important tissue and organ,mantle plays an irreplaceable role in the activities and shell formation of mollusks.In this study,the hard-shelled mussel was taken as the experimental object,and the roles of the three mantle folds in the shell periostracum formation and chitin related functions（such as synthesis,metabolism,binding）of mussels were explored by morphological observation and transcriptome analysis.The study is as follows:1.There were differences in the morphological and histological structures of the three mantle folds in M.coruscus by anatomical observation,paraffin sectioning and HE staining.The inner fold was the largest,followed by the middle fold and the outer fold was the smallest.There were also significant differences in the types of epithelial cells among the three folds.The outer fold at the periostracum groove had a simple columnar epithelium,in which the cells were slender,with a slender nucleus located at the base side.And there was a small amount of brown pigment in the cytoplasm.The cells in the simple columnar epithelium of middle fold（on the side near the periostracum）were short columnar with no pigment in the cytoplasm.However,a large amount of brown pigment could be observed in the epithelial cells of middle fold on the other side（far from the periostracum）.The inner fold had a simple columnar epithelium,with elongated nuclei located at the base of the epithelial cells.A large amount of dark brown pigment could be observed in the cytoplasm.2.In this study,powder solids were extracted from the outer periostracum layer and the inner shell layer using decalcification,depolymerization,and bleaching methods.The extracted products were identified using Fourier transform infrared spectroscopy（FTIR）,and characteristic chitin absorption peaks were found in these two extracted products.By comparing the FTIR absorption peaks,it was found that there were single absorption peaks at 3272 cm^-1and 3269 cm^-1,respectively.It was indicated that the chitin extracted from the periostracum layer and shell layer wereβ-Chitin.However,due to the differences in the composition of organic matter and minerals between the shell layer and the periostracum layer,the absorption peaks of the two chitin extracts shifted left and right.In addition,the chitin extract of shell layer had a smaller absorption peak at 1560 cm^-1,and the absorption peak of the periostracum layer at 1513 cm^-1～1626 cm^-1was also greater than that in the shell layer extract.It was suggested that the chitin content in the periostracum of hard-shelled mussel was higher than that in the shell layer.3.In this study,the three mantle folds（outer,middle and inner folds）transcriptome libraries were constructed and sequenced,and the differentially expressed genes among outer fold,middle fold and inner fold were analyzed.Based on the clustering analysis of the expression（FPKM）of three folds,it was found that the expression pattern of outer fold was significantly different from those of inner and middle folds.According to the transcriptome data,34 chitin related genes including chitin synthesis,degradation and binding functions were screened.By analyzing the domain and expression,it was found that chitinase（chitotriosidase）genes and chitin binding protein genes were mainly expressed in the outer fold,while chitin synthase genes and chitinase（beta-hexosaminidases and di-N-acetylchitobiase）genes were mainly expressed in the inner and middle folds.GO enrichment analysis of differentially expressed genes among the three folds also revealed that the inner and middle folds were mainly involved in the chitin biosynthesis,while the outer folds were mainly involved in regulating chitin binding,chitin metabolism,and chitin-containing cuticle pigmentation.This study explored the morphology of the three mantle folds,the chitin composition in the shell,and the transcriptome analysis of three folds.The transcriptome analysis revealed the different function of the three folds in chitin synthesis,chitin metabolism and chitin binding functions.It was suggested that the outer fold played a more important role in the formation of the cuticle and pigmentation of the mussel shell.This study will help to understand the chitin composition and chitin related genes in mussel shells,and provide theoretical reference for the research on shell formation and biomineralization.It provides a new theoretical basis for mussel cultivation and protection,and is of great significance in protecting and utilizing China’s marine resources and promoting the development of the marine fishery industry.