The molecular cloning and functional characterization of GLWamides neuropeptide in the adult and larvae of a stony coral Euphyllia ancora

• Main Authors: Yao, I-Chen, 姚毅前
• Other Authors: Chang, Ching-Fong
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/w38858

碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 106 === Neuropeptides are short bioactive peptides that act as modulators and hormones in various biological processes of animals. Previous studies showed that the exogenous treatment of planula larvae with a peptide identified in Hydra (Hym-248, EPLPIGLWamide), which belong to GLWamide (Glycine-Leucine-Tryptophan-NH2) family, has been shown to induce the larval settlement and metamorphosis of some Acropora corals. However, identification of the genes encoding any neuropeptides has not been reported in corals so far. Moreover, no study has clearly shown the GLWamide neurons in coral bodies at both embryonic and adult stages. Basic information regarding the nervous system in coral, such as morphology and spatial distribution of neuronal cells, and physiological function of neuropeptides, are still scarce. To get a better understanding of the nervous system in corals, we selected a stony coral Euphyllia ancora as the experimental animal and performed following experiments: (1) Identification of a cDNA encoding GLWamide preprohormone ; (2) Investigation of the spatial distribution of EaGLWamide preprohormone transcripts in the adult coral by qPCR; (3) Investigation of the distribution of GLWamide neurons in the adult and larvae by immunohistochemical analysis or whole mount-immunodetection with anti-GLWamide antibody; and (4) Establishment of the assay to investigate the putative function of GLWamides in the coral bodies. From the transcriptome database of E. ancora, a partial sequence exhibiting high similarity to GLWamide preprohormone of other cnidarians were identified. The full-length of GLWamide preprohormone cDNA in E. ancora (named EaGLW preprohormone) were successfully elucidated by 5’ and 3’ RACE-PCR. In the adult coral, a quantitative RT-PCR analysis showed that EaGLW preprohormone transcripts were highly expressed in the mouth and tentacle compared with other tissues in both sexes polyps. Immunohistochemical analysis with anti-GLWamide antibody demonstrated that GLWamide-neurons were mainly distributed in the ectodermal regions of the mouth and tentacle. Exogenous treatment of the isolated mouth and tentacles with GLWamide treatment was shown to induce the contraction of these tissues. Exogenous treatment of E. ancora polyp with GLWamides was shown to induce the polyp contraction. By contrast, in E. ancora embryos, quantitative-RT-PCR analysis revealed that EaGLW preprohormone transcripts were highly expressed in E. ancora embryo at 64 hr post-fertilization, which corresponds to the planula stage. Immunohistochemical analysis of the planula with the anti-GLWamide antibody revealed that GLWamide neurons were localized at the epidermis of the posterior region. Furthermore, treatment of planula larvae with GLWamides showed that the swimming speed of planula larvae became faster. The GLWamides treatment did not induce the settlement and metamorphosis of E. ancora planula. These results suggested that the GLWamides is involved in tissue contraction (myoactivity) of the adult E. ancora and the swimming activity in planula larvae. To the best of our knowledge, this study is the first to show the presence of the GLWamide preprohormone gene, the spatial distribution patterns of GLWamide neurons, and the putative function of GLWamide in the adult and larvae of stony corals. The data obtained in the present study will be valuable not only for better understanding of the neuronal system of stony corals, but also for the comparative evolutionarily biology of nervous system in metazoans.