Abstract
The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts.
Highlights
It is well known that the barnacle Balanus amphitrite is a major fouling marine animal and is widespread throughout the world
Characterization of Bar-p38 Mitogen activated protein kinase (MAPK) Based on the partial sequence from the barnacle transcriptome database and sequences of the 39 and 59 rapid amplification of cDNA ends (RACE) products, the fulllength coding region of p38 MAPK was obtained from the barnacle cDNA and was named Bar-p38 MAPK, with 363 deduced amino acids
The bioassay with the p38 MAPK inhibitor indicated the involvement of p38 MAPK in larval settlement
Summary
It is well known that the barnacle Balanus amphitrite is a major fouling marine animal and is widespread throughout the world. Before transformation from the planktonic to the sessile phase, cyprids actively explore the surrounding environment with their paired antennules to search for a suitable site for settlement and metamorphosis. Though they exist in nauplii, the antennules have been highly modified as attachment organs at the cyprid stage. Cyprids detect and examine the substrata for their chemical and physical natures with their antennules that are enriched with neuronal fibers. They respond and decide whether to settle or not.
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