| The veined rapa whelk(Rapana venosa),a typical carnivorous gastropod,widely distributed around the Bohai Sea,Yellow Sea and East China Sea,is an important economically shellfish in China.Metamorphosis is an important developmental process of R.venosa,the key of artificial breeding,and is directly related to the population dynamics and spatial expansion of R.venosa.Food habit transition is an important character of metamorphosis in R.venosa,which can ensure the success of metamorphosis.Therefore,the research on the food habit transition in R.venosa during metamorphosis is of great significance to reveal the regulation mechanism of larval metamorphosis,to promote the development of artificial breeding industry and to restore natural resources.In this study,we used RACE,Real Time PCR,16 S rRNA,ELISA,and LC-MS and combined with pharmacological experiments,to study the characteristics of food habit transition and its regulation mechanism in the metamorphosis of R.venosa.We revealed the characteristics of food habit transition of R.venosa and the regulation mechanism of metamorphosis induced by juvenile oyster from the digestive system and neuroendocrine system.The full-length c DNA sequences of key digestive enzymes and their expression characteristics in the whole early developmental stage were obtained,which further clarified the process of food habit transition;and screened the keystone taxa in symbiotic microbiota of R.venosa,and investigated the potential biological function;obtained the full-length c DNA sequence of the CCK receptor,which can regulate the key genes in food habit transition and metamorphosis of R.venosa.Additionally,by investigating the induction of juvenile oyster on the competent larvae of R.venosa,the effects of oyster induction on the digestive system and neuorendocrine system were identified,and the keystone taxa of symbiotic microbiota were screened,and the effects of juvenile oyster on the food habit transition and metamorphosis of R.venosa were analyzed by metabonomics.The main findings are as follows:1.Molecular cloning of key digestive enzyme genes and their expression analysis during early development stageThe full-length c DNA sequences of three key digestive enzyme genes(Trypsin,Carboxypeptidase and Cellulase)in the food habit transition and metamorphosis were obtained by RACE,and their Multiple sequence alignment and phylogenetic trees were constructed.Real Time PCR and enzyme activity analysis showed that the expression and activity of Trypsin and Carboxypeptidase increased significantly after metamorphosis,while the expression and activity of Cellulase decreased significantly after metamorphosis,it further explained the process of food habit transition.2.Characteristics of symbiotic microbiota in R.venosa during early development stageThe symbiotic microbiota during the development of R.venosa were studied using16 S rRNA high-throughput sequencing technique,1744 OTUs were identified and 32 phyla and 580 genera were taxonomic annotated.The core flora and dominant flora in the early development stage of R.venosa were identified by OTUs shared between groups,and Proteobacteria was the first dominant microflora.Based on the KEGG pathway enrichment analysis,the function of the symbiotic microorganisms was predicted,which were mainly enriched in the related functional pathways such as transport protein,purine metabolism and pyrimidine metabolism;and five key species which might be related to the early development stage of R.venosa were selected by correlation analysis,and the correlation analysis with digestive enzymes was carried out,the microbial populations associated with food habit transition were further screened,it was preliminarily determined that Rhodopathaceae and Flavobacteriaceae may be the key species in the process of food habit transition and metamorphosis.3.Cloning and expression of neourendocrine system key genes and their induction on R.venosaThe full-length c DNA of the CCK receptor gene was obtained by RACE,and the multiple sequence alignment and evolutionary tree were constructed.Analysis of Real Time PCR showed that CCK receptor expression increased significantly in V-Ⅳ stage,but significantly decreased after metamorphosis.CCK induced experiments showed that the expression of Carboxypeptidase was significantly increased induced by CCK,while the expression of Cellulase was significantly decreased,and the expression of 5-HT receptor and NOS were also significantly affected.In addition,the changes of other neurotransmitters and hormones related to digestion and ingestion in the metamorphosis and food habit transition were detected by ELISA,these results suggest that neuroendocrine system may play an important role in regulation of larval food batit transition and metamorphosis.4.Responses of larvae to the induction of juvenile oysterThe induction of juvenile oyster on the competent larvae of R.venosa was investigated by analyzing the changes in the expression of key digestive enzymes and neuroendocrine system critical genes,and the changes in the diversity,composition,structure and function of the symbiotic microbiota of the larvae,the changes of the larval metabolome and the association analysis of the symbiotic microbiota and the metabolome of the larvae,and revealed that the induction of juvenile oyster was similar to that of CCK,and make the expression of carboxypeptidase up-regulate significantly,while make he expression of cellulase down-regulate,indicating the effect of CCK on food habit transition.In addition,the expression of CCK receptor,5-HT receptor and NOS were also significantly affected,indicating the induction on the metamorphosis of larvae.5-HT was significantly up-regulated in the juvenile oyster-induced group,and the related signal transduction pathways were significantly enriched,suggesting that 5-HT and 5-HT receptor may mediate the metamorphosis induced by the juvenile oyster. |
PDF Full Text Request