IGFBP7Effects On Larval Settlement And Metamorphosis And Adult Immunoreactions In The Small Abalone Haliotis Diversicolor

Small abalone is a commercially important marine aquaculture mollusk species in severalsouthern provinces of China. It is also one of an ideal model for studying metamorphosis inmollusk and even marine invertebrates because abalone larvae are lecithotrophic and have abiphasic life-cycle that includes planktotrophic, settlement and metamorphosis. Up to now,some signal pathways of metamorphosis have been developed, but there is little knowledge ofmolecular mechanism regulating abalone larvae metamorphosis. Therefore, cloning andcharacterization of metamorphosis-related genes from small abalone by molecular biologicaltechniques will provide insights into the molecular mechanisms of abalone and even molluskmetamorphic development.Partial sequence of IGFBP7was obtained from our EST database of small abalone.Full-length cDNA of IGFBP7was cloned by SMART RACE technique and denoted assaIGFBP7. Real-time quantitative PCR (RT-PCR), whole mount in situ hybridization(WMISH), whole mount immunohistochemistry (WMICH), Western blot, and gene silencingand gene overexpression were used to reveal the role of saIGFBP7in abalone larval settlementand metamorphosis and in adult immunoreactions. The results were reported as follows:1) The full-length cDNA of saIGFBP7is1812bp, and the complete open reading frame of720bp encodes a protein of239amino acids. Analysis of conserved domains revealedsaIGFBP7includes the presence of an IGFBP N-terminal domain (IB), a kazal-type serineproteinase inhibitor domain (KI), and an immunoglobulin-like C2domain (IgC2). Additionally,two IGFBP7characteristic motifs were identified from the putative amino acid sequence ofsaIGFBP7: IB domain signature (xCGCCxxC) and KI domain signature (CGxDxxTYxN).Moreover,18conserved cysteine residues,2potential N-glycosylation sites and11proteinkinase C phosphorylation sites were also observed in the deduced amino acid sequence ofsaIGFBP7.2) The tissue distribution of saIGFBP7mRNA and protein was investigated by usingqRT-PCR and Western blot. The saIGFBP7mRNA and protein could be detected in allexamined tissues, with the highest expression level in hemocytes, higher expression level ingills (P<0.01). 3) saIGFBP7mRNA transcripts were observed in a subset of the branchial epithelium andthe nucleus of hemocytes by using the WMISH method. saIGFBP7protein was detected mainlyin the goblet-like cell of the branchial epithelium by WMICH.4) The saIGFBP7expression level at6h,12h after Vibrio parahaemolyticus challenge wasup-regulated significantly (P<0.05) in hemocytes. It was also quickly up-regulated at3h(P<0.05) in gill. The western blot analysis showed that the saIGFBP7protein was stronglyexpressed in gills at3h (P<0.05).5) qRT-PCR showed that saIGFBP7mRNA transcripts were rapidly increased in earlytrochophore stage, and constitutively highly expressed with the larval development (P<0.01).Furthermore, a gradually enhanced hybridization signal was predominantly restricted to thevicinity of the head and foot. saIGFBP7protein presented a similar distribution pattern as thatof WMISH, especially in the vicinity of the cilia.6) RNAi was employed to diminish the expression of endogenous saIGFBP7in theswimming-crawling late veliger. The statistical analysis showed that the saIGFBP7dsRNAinterference and adding insulin induced a31.81%and61.33%decrease (P<0.01) in larvalsettlement compared with the non-treated larvae, respectively.7) qRT-PCR, WMISH and WMICH was utilized to investigate the effectiveness of RNAiin the intrinsic saIGFBP7expression. The results showed that it was significantly suppressed bysaIGFBP7RNAi (P<0.01).8) Statistical analysis of the number of adherent cells showed that the inhibition ofsaIGFBP7in hemocytes did reduce cell adhesion (P<0.05), and the cell growth wassignificantly inhibited (P<0.01). Nevertheless, overexpression saIGFBP7stimulated thehemocytes proliferation and growth significantly for2days (P<0.01). Additionally, the westernblot was also consistent with our above findings.9）The possible molecular mechanisms underlying the developmental defects caused byloss of saIGFBP7was investigated, the results showed that saEGFL6and saVDAC2mRNAwas significantly up-regulated in swimming and creeping larval stage after saIGFBP7RNAi.saEGFL6had an enhanced hybridization signal restricted chiefly to the vicinity of the visceralmass and foot. Furthermore, saEGFL6had an enhanced hybridization signal restricted chieflyto the vicinity of the visceral mass.10）The full-length cDNA of saEGFL6is1460bp. The complete open reading frame of1053bp encodes a protein of350amino acids. Analysis of conserved domains revealed thatsaEGFL6includes three epidermal growth factor-like domains (EGF) and two EGF-likedomains. Moreover,3potential N-glycosylation sites and22protein kinase C phosphorylationsites were observed in the deduced amino acid sequence of saEGFL6. 11) qRT-PCR was employed to investigate the tissue distribution of saEGFL6andsaVDAC2mRNA. The saEGFL6and saVDAC2mRNA could be detected in all examinedtissues. The saEGFL6gene had the highest expression level in hemocytes, higher expressionlevel in hepatopancreas, mucous glands, and epipodiums (P<0.01). Furthermore, the saVDAC2gene had the the lowest expression level in hemocytes, highest expression level in kidney,higher expression level in hepatopancreas and mantle (P<0.01).12) The temporal expression patterns of saVDAC and saEGFL6of different developmentalstages were analyzed by qRT-PCR, respectively. The results showed that the saVDAC mRNAexpression level was quickly increased in trochophore stages (P<0.01) and declinedsignificantly in veliger (P<0.01).The copy numbers of saEGFL6mRNA transcripts werequickly and significantly increased in late trochophore and early veliger stages (P<0.01).