Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite

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 Ca(2+)/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.

Figure 1. The complete nucleotide and deduced amino acid sequences of CaM in B. amphitrite.

The nucleotides and amino acids are numbered on the left of the sequences. The sequences in the box show the whole open reading frame. An asterisk (*) represents the stop codon at the end of the coding sequence.

Figure 2. Multiple alignment of the amino acid sequence of CaM in B. amphitrite with its homologs in sponge (BAB61797.1), sea anemone (BAB61796.1), sea slug (NP_001191509.1), copepod (ACO10440.1), sea urchin (BAB89360.1), ascidian (NP_001027633.1), hagfish (AAD56955.1), frog (NP_001080864.1), bovine (NP_001159980.1), mouse (AAH54805.1), and human (AAD45181.1).

Amino acids are numbered and asterisks (*) indicate the identical amino acids. The four EF-hand domains are underlined and the letters in boxes show the conserved tyrosine (Y) residues generally found in most invertebrates. The numbers above the sequences show the amino acid position. The shaded areas are EF-hand domains.

Figure 3. Southern blot of digested genomic DNA for the identification of the copy number of Ba-CaM gene.

Genomic DNA was digested with various restriction enzymes including BamHI, BglII, EcoRI, HindIII, NcoI, and SspI.

Figure 4. Ba-CaM mRNA expression levels in different developmental stages of B. amphitrite detected by real-time PCR.

The stages included stage VI nauplius (nau6), cyprid (cyp) and juvenile (juv). Bars represent the mean ± SD (n = 3). An Asterisk (*) indicates a significant difference compared with the positive control (P<0.05).

Figure 5. Ba-CaM spatial expression pattern in the sagittal sections of B. amphitrite cyprids.

The HE staining image (A) shows a clear larval histology. Blue color stained by haematoxylin indicated nuclei, while red color by eosin indicated basic proteins or muscle fibers. Lenses of compound eyes and intracellular substances (maybe cement proteins) in the cement gland were stained by eosin. Positive signals were detected from the sections hybridized with the anti-sense probe (B). Section hybridized with the sense probe served as a control (C). (E) Detail of the cyprid compound eye and cement glands. (F) Detail of the cyprid posterior ganglion. Ce, compound eye; Cg, cement gland; Pg, posterior ganglion; Ta, thorax. Scale bars = 100 µm.

Figure 6. The effect of inhibitors on larval settlement of B. amphitrite.

Inhibitor assays using (A) the CaM inhibitor compound 48/80, (B) the MLCK inhibitor ML-7, (C) the CaMKII inhibitor KN-62, and (D) the CaMKII inhibitor AIP. Data presented are the means ± SD (n = 5). Asterisks (*) indicate significant differences compared with the positive control (P<0.05).

Figure 7. Ba-MLCK and Ba-CaMKII mRNA expression levels in different developmental stages of B. amphitrite.

Relative gene expression levels of MLCK (A) and CaMKII (B) were detected by using real-time PCR in stage VI nauplius (nau6), cyprid (cyp) and juvenile (juv). Bars represent the mean ± SD (n = 3). Asterisks (*) indicate significant differences compared with the positive control (P<0.05).