Inhibitory function of nitric oxide on the onset of metamorphosis in competent larvae of Crepidula fornicata: A transcriptional perspective

In diverse invertebrate species characterized by a biphasic life cycle, metamorphosis represents a fundamental biological transition which determines the fate of benthic population dynamics through settlement and recruitment. Within this context, nitric oxide (NO) is thought to act as an endogenous inhibitor of metamorphosis. While attention has been focused on the mechanisms of this inhibitory pathway with pharmacological agents and immunohistochemistry tools, relatively few studies have investigated transcriptional process at the origin of NO synthesis. In this paper, we report the isolation of a 218-bp cDNA fragment of an ortholog of the neuronal nitric oxide synthase (nNOS) gene in the invasive marine species Crepidula fornicata. By the use of quantitative real-time PCR, we examine the transcription of this gene throughout larval development and in response to two inducers [K+ excess (20 mM), dibromomethane (DBM) (1000 ppm)] that are known to potentiate metamorphosis in invertebrate species. The level of transcription increased constantly during the larval development, suggesting an increased repressive effect over metamorphosis as larvae aged. Interestingly, maximum values were reached 6 h post-treatment, before declining within 20 h for all the tested conditions. Overall, our results are in agreement with the involvement, at a molecular level, of the NO signalling pathway in metamorphosis. The decrease in nNOS gene transcription post-induction could support the inhibitory effect of NO upon the onset of metamorphosis in competent larvae, although further studies are needed to fully describe the pathways triggered by K+ ions and DBM induction. Furthermore, results indicate that metamorphosis could occur after termination of endogenous inhibition, bringing support to the hypothesis of spontaneous metamorphosis.