| Metamorphosis is the essential stage during ontogenesis of several important aquaculture animals such as A. japonicus, covering a wide range of significant changes in the morphology and ecological habit of larvae. caspase-8 has been confirmed to be the foremost apoptotic promoter, which participates in embryonic development and immune defense processes in many aquatic animals such as fish and shellfish. Aiming to identify the role of caspase-8 gene in larval growth of sea cucumber A. japonicus, RACE technology was used to obtain its full-length cDNA with the following analyses on sequence characteristics; the Ajcaspase-8expression levels were detected in adult tissues and larval development periods using real-time fluorescence quantitative PCR method; the expression location of Ajcaspase-8 and cell apoptosis in metamorphosizing larvae were presented by whole embryo hybridization and TUNEL techniques, respectively. The results indicated that the full-length cDNA of Ajcaspase-8 gene was 2790bp, which encoded 671 amino acids containing hydrolysis active CASc domain and the conserved pentapeptide QACQG of Caspase family. Moreover, death receptor domain (DED) and two subunits (P20 and P10) were present in Ajcaspase-8 deduced protein, acting as the key motifs to perform the function of an initiator caspase in apoptosis pathway. Ajcaspase-8 was clustered into a branch with Holothuria leucospilota, occupying closer genetic distance to Oryzias latipes, which showed an advanced evolutionary status of this molecule. RT-qPCR results showed that the Ajcaspase-8 gene in adult sea cucumbers exhibited housekeeping expression, with the highest expression in coelomocyte and the lowest in gonads (P<0.05), indicating that it is involved in immunity. During the whole development process of A. japonicus larvae, the expression of this gene was all detected, with the top value in doliolaria stage and second-highest in juvenile stage (P<0.05), while both significantly higher than those of other developmental stages (P<0.05). This finding suggests that Ajcaspase-8 had a potential role in the morphological reshaping of larvae during metamorphosis. The whole in situ hybridization analysis exhibited that the Ajcaspase-8 positive signals were only localized in the stomach of late-stage auricularia, concentrated near the hyaline sphere of doliolaria, and obviously intensified in the intestine of juveniles, indicating this gene was actively expressed in the redundant and digestive tissues of larvae during metamorphosis. TUNEL visualization showed that the number of apoptotic cells had an increasing trend along with metamorphosis, hinting that apoptosis was an important way to maintain homeostasis of metamorphic larvae. Comprehensively, these above-mentioned results showed that the Ajcaspase-8 gene is likely to play important roles in the metamorphosis and attachment of A. japonicus, speculating that it could initiate cell apoptosis to participate in the morphological reconstruction and immune defense during the crucial process in ontogenesis of this species. This study provides fundamental data for further investigating the regulatory mechanisms of metamorphosis in sea cucumbers. |
