| Pearl oyster Pinctada fucata martensii,mainly distributed in tropical and subtropical sea areas,is the main species of seawater pearls cultivated in China.The species is framed in Guangdong,Guangxi and Hainan,China.The metamorphic is the stage of transition from planktonic larval stage to adult bivalve and is the most important stage in the life history of bivalve,which has a direct impact on the population change.The study of mechanism of metamorphosis and larval growth and development pattern of the pearl oyster is of great practical and theoretical significance to promote the work of breeding,resource recovery,and to reveal the population change and distribution pattern in natural marine areas.In this study,we investigated the mechanism on metamorphosis of pearl oyster larvae by a combination of RNA-seq and LC/MS techniques.The regulatory features of the metamorphosis process of larvae were revealed at the transcriptome and metabolome levels.The key transcripts and metabolites related to neurotransmitters in the metamorphosis of larvae of the pearl oyster were obtained,and their potential biological functions in the metamorphosis of the pearl oyster were investigated.We also investigated the effects of different metabolites on the settlement of larvae through the treatment of the larvae with different metabolites from metabolism.We obtained c DNA sequences of key genes 5-HTR2-like and 5-HTR4-like and investigated the expression pattern of three 5-HT receptors during the metamorphosis of the pearl oyster.1.RNA-seq technique was used to investigate the gene expression pattern in three different stages of the pearl oyster to construct a network of important functional genes related to the developmental regulation of Pinctada fucata martensii,and to systematically propose a molecular mechanisms regulating the larval metamorphosis.We also found that the dopaminergic pathway,serotonergic pathway,GABA pathway and glutamatergic pathway were significantly involved in the metamorphosis of Pinctada fucata martensii by KEGG enrichment analysis.2.We conducted the metabolomics data during metamorphosis of larvae of the pearl oysterby using LC-MS technique,and identified 633（positive mode）and 433（negative mode）differential metabolites between the eye-spot larvae and settled larvae,and 660（positive mode）and 382（negative mode）differential metabolites between settled larvae and juvenile.Among the differential metabolites of metamorphosis,dopamine,epinephrine,norepinephrine,serotonin,GABA and c AMP were hypothesized to be involved in the settlement and metamorphosis of Pinctada fucatamartensii.The metabolic pathway analysis revealed that the metabolic pathways of phenylalanine,tyrosine and tryptophan biosynthesis,ubiquinone and other terpenoid quinone biosynthesis,D-glutamate and D-glutamine metabolism,arachidonic acid metabolism and sterol hormone biosynthesis played important roles in the metamorphosis of Pinctada fucata martensii.Serotonin,GABA,ecdysteroid,adenine nucleotide,hypoxanthine nucleotide,hypoxanthine,glutamate,NMDA and flupirtine maleate was selected for further experiments.3.By treating larvae of Pinctada fucata martensii with metabolites from the second part,it was found that 5-hydroxytryptamine was the most effective among all the inducers in improving larval attachment,with a 6.7-fold increase compared to the control group.GABA was also effective,with a 4.6-fold increase compared to the control group.Ecdysone was the most effective in treating larvae at 10-5 M for 72 hours,with a 1.6-fold increase compared to the control group.All purines were induced on larvae after 48h,and the maximum increase rate ranged from 0.76 to 2.5.Glutamate was the most effective at 10-4M for 12 h with 2.8-fold increase in settlement rate.Flupirtine maleate,an NMDA receptor blocker,was able to increase the settlement rate by 1-fold at 10-6 M for 48 h treatment of larvae.4.The full length of the c DNA of 5-HTR2-like and 5-HTR4-like genes,which are key genes in the larval metamorphosis,were cloned and obtained for the first time in Pinctada fucata martensii by RACE technique.5-HTR2-like was 1,650bp,encoding469 amino acids with a typical 7 transmembrane domain;5-HTR4-like was 2,118 bp,encoding 394 amino acids with a typical 7 transmembrane structural domain.A phylogenetic tree of 5-HT receptor was constructed.The results of fluorescence quantitative PCR revealed that the 5-HTR2-like receptor was found to be highly expressed in the eyed larval stage,but its expression was significantly down-regulated after the settlement;the 5-HTR4-like receptor was lowly expressed in the eyed larval stage,but its expression was significantly up-regulated after settlement.The early expression patterns and localization of 5-HTR-1like,5-HTR2-like and 5-HTR4-like genes in the larvae of the pearl oyster were revealed by in situ hybridization. |
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