Involvement of GLWamide neuropeptides in polyp contraction of the adult stony coral Euphyllia ancora

International audienceThe existence and function of neurons remain largely unexplored in scleractinian corals. To gain a better understanding of neuronal functions in coral physiology, this study focused on Glycine-Leucine-Tryptophan-amide family neuropeptides (GLWamides), which have been shown to induce muscle contraction and larval metamorphosis in other cnidarians. Molecular identification and functional characterization of GLWamides in the adult stony coral Euphyllia ancora were performed. We successfully elucidated the full-length cDNA of GLWamide preprohormone in E. ancora (named EaGLW preprohormone). The deduced amino acid sequence was predicted to contain six potential GLWamide peptides. Tissue distribution analysis demonstrated that transcripts of EaGLW preprohormone were mainly expressed in the mouth (including the pharynx) and tentacles of the polyps. Immunodetection with an anti-GLWamide monoclonal antibody revealed that GLWamide neurons were mainly distributed in the epidermis of the mouth region and tentacle, in agreement with the distribution patterns of the transcripts. Treatment of the isolated mouth and tentacles with synthetic GLWamide peptides induced the contraction of these isolated tissues. Treatment of polyps with synthetic GLWamide peptides induced the contraction of polyps. These results suggest that GLWamides are involved in polyp contraction (myoactivity) in adult scleractinians. Our data provide new information on the physiological function of neuropeptides in scleractinians

Characterization of anti-Eavas immunoreactivity in tentacle and mesenterial filament.

<p><b>A</b>. irEavas-positive cells in the tentacle region. <b>B</b>. Higher magnification views of the insets shown in <b>A</b>. The arrows indicate irEavas-positive cells. <b>C</b>. irEavas-positive cells from a mesenterial filament. <b>D</b>. Higher magnification views of the insets shown in <b>C</b>. The scale bars represent 50 µm in panels <b>A</b> and <b>C</b> and 20 µm in panels <b>B</b> and <b>D</b>. m, mesoglea; ect, ectoderm; end, endoderm.</p

Expression of Eavas mRNA and protein in unfertilized mature eggs of <i>E. ancora</i>.

<p><b>A</b>. RT-PCR analysis of <i>Eavas</i> expression in unfertilized mature eggs. The unfertilized mature eggs that were spawned in aquaria were collected from three different colonies, shown as Sample 1, Sample 2, and Sample 3. β-Actin was used as a positive control for the PCR reaction. Reactions lacking either the reverse transcriptase (RT negative) or template (N.C.) were included as negative controls for each set of reactions. <b>B</b>. Western blotting analysis of the anti-Eavas antibody. Protein extracts prepared from unfertilized mature eggs (12.5 µg) were separated by SDS-PAGE. After transferring to a nitrocellulose membrane, the proteins were immunoblotted with the anti-Eavas antibody (anti-Eavas) or anti-β-actin antibody (anti-β-actin). The unfertilized mature eggs that were spawned in aquaria were collected from three different colonies, shown as Sample 1, Sample 2, and Sample 3. The molecular weight markers are shown in the right.</p

Tissue distribution of <i>Eavas</i> mRNAs and characterization of the anti-Eavas antibody.

<p><b>A</b>. The tissue distribution of <i>Eavas</i> transcripts was determined by semiquantitative RT-PCR analysis of male and female coral samples collected in Februay 2011. The samples examined include whole tissue, isolated tentacles, and the mesentery, the latter of which encompasses the gonad. β-Actin was used as the internal control. Reactions lacking either the reverse transcriptase (RT−) or template (N.C.) were included as negative controls for each set of reactions. <b>B</b>. Western blotting analysis of the anti-Eavas antibody. Protein extracts prepared from a Februay 2011 male sample (12.5 µg) were separated by SDS-PAGE. After transferring to a nitrocellulose membrane, the proteins were immunoblotted with the anti-Eavas antibody (anti-Eavas) or the anti-Eavas antibody preadsorbed with the peptide antigen (preadsorbed). The molecular weight markers are shown in the middle.</p