Abstract
Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.
Highlights
Barnacles are a major sessile component in intertidal ecosystems around the world, and a dominant hard fouler of ship hulls and other marine structures [1]
Many previous studies have focused on the involvement of endogenous factors during barnacle larval attachment and metamorphosis in the cosmopolitan intertidal barnacle Balanus amphitrite, such as cyclic AMP [5], protein kinase C [6], settlement-inducing protein complex (SIPC) [7], and hormonal substances [8,9,10]
The blastn result revealed that the coding region of Barnacle CaM gene (Ba-CaM) matched the CaMs from copepod (79%), sea slug (77%), ascidian (78%), sea urchin (83%), hagfish (80%), frog (77%), rat (78%), and human (74%)
Summary
Barnacles are a major sessile component in intertidal ecosystems around the world, and a dominant hard fouler of ship hulls and other marine structures [1]. During metamorphosis into the juvenile, several morphological changes occur, including the decortication of cyprid carapace, the formation of a new chitinous layer, the degeneration of compound eyes and antenna, the migration of naupliar eye, and the development of feeding cirri [3]. This transition from free-swimming larvae to sessile juveniles is crucial for their development, and for species distribution and intertidal community dynamics [4]. More molecular studies have been conducted in recent years to detect the differential gene and protein expression profiles during larval settlement of this species. The CaM protein product first accumulated in the stage VI nauplius stage and peaked in the cyprid stage, followed by a decline in attached larvae and juveniles [14]
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