Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the ‘aragonitic line’. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P.fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the ‘aragonitic line’, and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.

Molluscs form their shells out of CaCO₃ and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO₃ were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

Bee venom has been reported to exhibit antitumour activity in‐vitro and in‐vivo. Apoptosis, necrosis and lysis of tumour cells were suggested as possible mechanisms by which bee venom inhibited tumour growth. The aim of this study was to investigate potential mechanisms by which bee venom inhibits K1735M2 mouse melanoma cells in‐vitro and B16 melanoma, a transplantable solid melanoma in C57BL/6 mice, in‐vivo. The proliferation of K1735M2 cells in‐vitro was inhibited by bee venom in a concentration‐ and time‐dependent manner. The inhibition was indicated by the arrest of the cell cycle at the G1 stage, as detected by flow cytometric measurements. The bee venom induced apoptosis‐like cell death as identified by histological observations and by DNA fragmentation. In the in‐vivo experiments, the bee venom (1.0, 3.0, 9.0 mg kg⁻¹ of body weight, on days 1–12) was injected intraperitoneally into mice 24 h after the mice were inoculated with B16 cells. Inhibition of the solid tumour was observed. Apoptosis of the K1735M2 cells was suggested as the possible mechanism by which bee venom inhibited cell proliferation and induced K1735M2 cell differentiation in‐vitro. The in‐vivo experiment indicated that bee venom could be used as a chemotherapeutic agent against malignant tumours.

Ocean acidification and global warming have been shown to significantly affect the physiological performances of marine calcifiers; however, the underlying mechanisms remain poorly understood. In this study, the transcriptome and biomineralization responses ofPinctada fucatato elevated CO₂(pH 7.8 and pH 7.5) and temperature (25 °C and 31 °C) are investigated. Increases in CO₂and temperature induced significant changes in gene expression, alkaline phosphatase activity, net calcification rates and relative calcium content, whereas no changes are observed in the shell ultrastructure. “Ion and acid-base regulation” related genes and “amino acid metabolism” pathway respond to the elevated CO₂(pH 7.8), suggesting thatP. fucataimplements a compensatory acid-base mechanism to mitigate the effects of low pH. Additionally, “anti-oxidation”-related genes and “Toll-like receptor signaling”, “arachidonic acid metabolism”, “lysosome” and “other glycan degradation” pathways exhibited responses to elevated temperature (25 °C and 31 °C), suggesting thatP. fucatautilizes anti-oxidative and lysosome strategies to alleviate the effects of temperature stress. These responses are energy-consuming processes, which can lead to a decrease in biomineralization capacity. This study therefore is important for understanding the mechanisms by which pearl oysters respond to changing environments and predicting the effects of global climate change on pearl aquaculture.