Figure 1.
Maps of the sampling location.
The maps are showing the sampling location in Nanwan Bay at the southern coast of Taiwan.
Figure 2.
Photographic and schematic representation of the body structure of E. ancora.
A. E. ancora in an aquarium. The brown color in the tentacles is from Symbiodinium sp. t, tentacle; s, skeleton. B. Schematic of E. ancora body structure. C. Schematic of E. ancora horizontally dissected at the point indicated in B. The sites of gametogenesis are located between the retractor muscle and mesentery filament in the mesentery.
Figure 3.
Reproductive cycle of E. ancora in both female and male according to histological analysis by H & E staining.
A. Periods of male and female gametogenesis and dates of mass spawning between May 2010 and May 2011. B–F. Stages of oogenesis. B. Stage I. The arrow points to an oocyte. C, stage II; D, stage III; E, stage IV; and F, stage V. The arrow indicates a germinal vesicle that appears ‘U’-shaped due to the indentation of the plasma membrane. G–K. Stages of spermatogenesis. G. stage I. H, stage II; I, stage III; J, stage IV; and K, stage V. The broken lines indicate male germ cell clusters. Sg, spermatogonia; Sc I, primary spermatocytes; Sc II, secondary spermatocytes; St, spermatids; S, sperms. The scale bars represent 20 µm in panels B and G–K and 50 µm in panels C–F. The stage classifications are referred to Table 1. Gametogenesis frequency distribution for male and female E. ancora sampled between May 2010 and May 2011. L. The frequency distribution of oogenic stages among female corals (n = 3 colonies). Stage classification was performed according to the criteria provided in Table 1. M. Temporal changes in mean diameter of oocytes. The data are reported as the mean ± SEM (June 8, n = 30 eggs; August 6, n = 64 eggs; October 10, n = 88 eggs; December 5, n = 298 eggs; January 10, n = 236 eggs; February 14, n = 306 eggs; March 15, n = 782 eggs; April 14, n = 519 eggs; May 18, n = 441 eggs). Significant differences (P<0.05) are indicated with the different lower case letters. N. The frequency distribution of spermatogenic stages among male corals (n = 3 colonies). Stage classification was performed according to the criteria provided in Table 1. O. Temporal changes in mean diameter of male germ cell cluster. The data are expressed as the mean ± SEM (Cluster number: December 5, n = 104; January 10, n = 102; February 14, n = 100; March 15, n = 102; April 14, n = 103; May 18, n = 111). Significant differences (P<0.05) are indicated with the different lower case letters. P. Monthly mean temperatures of the sampling sites.
Table 1.
Criteria for classification of stages in gonadal development, as observed in histological sections.
Figure 4.
The deduced protein sequence of Eavas and schematic of the Eavas domain structure.
A. The deduced Eavas amino acid sequence. *, motifs conserved among DEAD-box protein family members; black dotted line, CCHC zinc fingers; black box with dotted line, RGG motif; black box, location of the antigen sequence used for antibody production. B. Schematic figure depicting the Eavas domain structure and regions that are highly homologous among the Vasa subfamilies. Vertical lines mark the positions of the nine conserved DEAD-box motifs. C. A phylogenetic tree comparing the amino acid sequences of Vasa- and PL10-related proteins from various taxa. The region used for this analysis corresponds to the sequence AFLLPV…LDEA (from 276 to 385 residues of Eavas), where are available and comparable region among various taxa. The sequences were aligned by a multiple sequence alignment using MUSCLE. The phylogenetic tree was constructed using the neighbor-joining method. The number at each node represents the bootstrap probability (%); the branches shown correspond to values of 50% and higher. The names and corresponding GenBank accession numbers of the proteins analyzed are as follows: Acropora CnVas (Acropora digitifera, BAB13683), Euphyllia Eavas (Euphyllia ancora, JQ968407), Nematostella Nvvas1 (Nematostella vectensis, AAW29073), Nematostella Nvvas2 (Nematostella vectensis, AAW29074), Tima Cnvas1 (Tima Formosa, BAB13687), Hydractinia Cnvas (Hydractinia echinata, BAB13686), Hydra Cnvas1 (Hydra vulgaris, BAB13307), Hydra Cnvas2 (Hydra vulgaris, BAB13308), Ephydatia PoVAS1 (Ephydatia fluviatilis, BAB13310), Ciona Ci DEAD1 (Ciona intestinalis, BAA36710), Xenopus XVLG1 (Xenopus laevis, NP_001081728), Danio vasa (Danio rerio, AAI29276), Gallus Cvh (Gallus gallus, BAB12337), Rattus VLG (Rattus sp., AAB33364), Mus Mvh (Mus musculus, BAA03584), Schistocerca vasa-like (Schistocerca gregaria, AF510054), Drosophila vasa (Drosophila melanogaster, NP_723899), Bombyx BmVLG (Bombyx mori, BAA19572), Acropora CnPL10 (Acropora digitifera, BAB13676), Euphyllia EaPL10 (Euphyllia ancora, JQ968406), Nematostella NvPL10 (Nematostella vectensis, AAW29072), Hydractinia CnPL10 (Hydractinia echinata, BAB13679), Hydra CnPL10 (Hydra vulgaris, BAB13306), Ephydatia PoPL10 (Ephydatia fluviatilis, BAB13309), Danio pl10 (Danio rerio, NP_571016), Xenopus ddx3x (Xenopus laevis, NP_001080283), Mus PL10 (Mus musculus, AAA39942), Mus p68 (Mus musculus, CAA46581), and Saccharomyces p68 (Saccharomyces cerevisiae, CAA36874).
Figure 5.
Tissue distribution of Eavas mRNAs and characterization of the anti-Eavas antibody.
A. The tissue distribution of Eavas 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. 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.
Figure 6.
Characterization of anti-Eavas immunoreactivity in female colonies.
Immunohistochemical analysis with the anti-Eavas antibody was performed to examine Eavas immunoreactivity (irEavas) in cells at different developmental stages of oogenesis. A. irEavas-positive oogonia located along the mesoglea (arrowheads) of samples harvested in May, just 5 days after spawning. m, mesoglea. B. A higher magnification view of the inset shown in A. Arrows indicate oogonia that display irEavas staining. C. irEavas-positive oocytes in the mesentery of corals that were sampled in August (stages I–II). D. A higher magnification view of the inset shown in C. The arrows indicate oocytes that display irEavas staining. E. irEavas-positive oocytes in the mesentery of coral samples collected in October (stage II). F. irEavas-positive oocytes from a February coral sample (stages III–IV). G. An oocyte (dotted line) from a coral sample collected in April (stage V). The arrows indicate irEavas-positive nuclei. The scale bars correspond to 100 µm in panels A, C, and E–G and 10 µm in panels B and D.
Figure 7.
Characterization of anti-Eavas immunoreactivity in male colonies.
Immunohistochemical analysis with the anti-Eavas antibody was conducted to examine Eavas immunoreactivity (irEavas) in cells at different stages of spermatogenesis. A. irEavas-positive spermatogonia located along the mesenterial mesoglea of a coral sample harvested in August (stage 0). B. A higher magnification view of the inset shown in A. Arrowheads indicate spermatogonia that display darker Eavas staining than others. C. Spermatogonia located along the mesenterial mesoglea of a coral sample harvested in October (late stage 0). The arrows indicate small spermatogonial clusters. D. A higher magnification view of the inset shown in C. The arrow indicates a spermatogonial cluster. The arrowheads indicate spermatogonia that exhibit darker irEavas staining compared with other spermatogonia. E. irEavas-positive spermatogonia located in the mesentery of a coral sampled in December (stage I). The arrows indicate spermatogonial clusters. F. A higher magnification view of the inset shown in E. G. irEavas stained spermatocytes located in the mesentery of a coral sampled in March (stage II). The arrows indicate the clusters within the mesoglea. H. A higher magnification view of the inset shown in G. I. Immunohistochemical analysis with the anti-Eavas antibody to examine Eavas immunoreactivity in stage III and IV male germ cells that were collected in April. J. Immunohistochemical analysis of stage V male germ cells collected in May. III, stage III; IV, stage IV; V, stage V. K. Immunohistochemical characterization of the anti-Eavas antibody using stage I male germ cells. L. The anti-Eavas antibody was preadsorbed with the peptide antigen. The preadsorbed control sample was nearly devoid of immunoreactivity. The arrow points to a cluster of male germ cells. The scale bars correspond to 50 µm in panels A, C, E, G, I and J, and 10 µm in panels B, D, F, H, K and L.
Figure 8.
Characterization of anti-Eavas immunoreactivity in tentacle and mesenterial filament.
A. irEavas-positive cells in the tentacle region. B. Higher magnification views of the insets shown in A. The arrows indicate irEavas-positive cells. C. irEavas-positive cells from a mesenterial filament. D. Higher magnification views of the insets shown in C. The scale bars represent 50 µm in panels A and C and 20 µm in panels B and D. m, mesoglea; ect, ectoderm; end, endoderm.
Figure 9.
Expression of Eavas mRNA and protein in unfertilized mature eggs of E. ancora.
A. RT-PCR analysis of Eavas 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. 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.