Duplicated paralog of sulfide: quinone oxidoreductase contributes to the adaptation to hydrogen sulfide-rich environment in the hydrothermal vent crab, Xenograpsus testudinatus.

The hydrothermal crab, Xenograpsus testudinatus (xtcrab) inhabits shallow-water, hydrogen sulfide (H2S)-rich hydrothermal vent regions. Until now, the adaptative strategy of xtcrab to this toxic environment was unknown. Herein, we investigated the sulfide tolerance and detoxification mechanisms of xtcrabs collected in their high-sulfide hydrothermal vent habitat. Experimental immersion of xtcrab in various sulfide concentrations in the field or in aquaria assessed its high sulfide tolerance. HPLC measurement of hemolymph sulfur compounds highlighted xtcrab detoxification capacity via catabolism of sulfide into much less toxic thiosulfate. We focused on a key enzyme for H2S detoxification, sulfide: quinone oxidoreductase (SQR). Cloning and phylogenetic analysis revealed two SQR paralogs in xtcrab, that we named xtSQR1 and xtSQR2. As shown by qPCR, xtSQR2 and xtSQR1 were expressed in the digestive gland, suggesting the involvement of both paralogs in the detoxification of food-related H2S. In contrast, xtSQR1 transcript was highly expressed in the gill, while xtSQR2 was not detectable, suggesting a specific role of SQR1 in gill detoxification of H2S of environmental origin. Comparison between xtcrabs in their hydrogen sulfide-rich hydrothermal habitat, and xtcrabs maintained for one month in sulfide-free seawater aquarium, showed higher transcript levels of gill xtSQR1 in sulfide-rich habitat, further supporting the specific role of xtSQR1 paralog in environmental H2S detoxification in the gill. Gill SQR protein level as measured by Western blot, and gill SQR enzyme activity were also higher in sulfide-rich habitat. Immunohistochemical staining further showed that SQR expression was co-localized with Na+/K+-ATPase-positive epithelial and pillar cells of the gill filament. This is the first evidence of duplicate SQR genes in crustaceans. Overall, our study suggests that the subfunctionalization of duplicate xtSQR genes may play an important role in sulfide detoxification to maintain the sulfide homeostasis in X. testudinatus, providing an ecophysiological basis for its adaptation to the high-sulfide hydrothermal vent environment.

An Indo-Pacific coral spawning database.

The discovery of multi-species synchronous spawning of scleractinian corals on the Great Barrier Reef in the 1980s stimulated an extraordinary effort to document spawning times in other parts of the globe. Unfortunately, most of these data remain unpublished which limits our understanding of regional and global reproductive patterns. The Coral Spawning Database (CSD) collates much of these disparate data into a single place. The CSD includes 6178 observations (3085 of which were unpublished) of the time or day of spawning for over 300 scleractinian species in 61 genera from 101 sites in the Indo-Pacific. The goal of the CSD is to provide open access to coral spawning data to accelerate our understanding of coral reproductive biology and to provide a baseline against which to evaluate any future changes in reproductive phenology.

The plasticity of gonad development of sexual reproduction in a scleractinian coral, Porites lichen.

Metazoans have evolved a complexity of sexual system and gonad development, however, sexual reproduction of scleractinian corals is not well understood. This study aimed to address the sexual system and gametogenesis in Porites lichen, a common species in the Indo-West Pacific. This study represents the first description of sexual system, which were determined by histological analysis of the samples collected in northern Taiwan. In addition, female and hermaphroditic colonies were separately cultured in aquarium to further monitor the release of eggs/larvae and thereby confirm the breeding system. The results demonstrate that P. lichen is a polygamodioecious brooder and displays seasonal gametogenesis and embryogenesis that ends in late summer. In hermaphroditic colonies, male polyps are predominant and hermaphroditic polyps make up a very small percent (1%-19.3%). In addition, two new gametogenic features were observed from the histological analysis: 1) oocytes developed within the spermaries in hermaphroditic polyps during the early stage of gametogenesis and 2) melanin granular cells were clustered in spermaries in both male and hermaphroditic colonies. This study demonstrated the plasticity of gametogenesis and melanin related cells appeared in corals, which provides an important information to explore hormones and molecular mechanism involving in gonadal arrangement and production of melanin for further studies.

Discovery of a receptor guanylate cyclase expressed in the sperm flagella of stony corals.

The receptor guanylate cyclases (rGCs) in animals serve as sensitive chemoreceptors to detect both chemical and environmental cues. In reproduction, rGCs were shown to be expressed on sperm and serve as receptors for egg-derived sperm-activating and sperm-attracting factors in some echinoderms and mammals. However, sperm-associated rGCs have only been identified in some deuterostomes thus far, and it remains unclear how widely rGCs are utilized in metazoan reproduction. To address this issue, this study investigated the existence and expression of rGCs, particularly asking if rGCs are involved in the reproduction of a basal metazoan, phylum Cnidaria, using the stony coral Euphyllia ancora. Six paralogous rGCs were identified from a transcriptome database of E. ancora, and one of the rGCs, GC-A, was shown to be specifically expressed in the testis. Immunohistochemical analyses demonstrated that E. ancora GC-A protein was expressed in the spermatocytes and spermatids and eventually congregated on the sperm flagella during spermatogenesis. These findings suggest that GC-A may be involved in the regulation of sperm activity and/or functions (e.g., fertilization) in corals. This study is the first to perform molecular characterization of rGCs in cnidarians and provides evidence for the possible involvement of rGCs in the reproduction of basal metazoans.

Immunodetection of acetylated alpha-tubulin in stony corals: Evidence for the existence of flagella in coral male germ cells.

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies.

Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

A Novel Female-Specific and Sexual Reproduction-Associated Dmrt Gene Discovered in the Stony Coral, Euphyllia ancora.

Transcription factors encoded by the Dmrt gene family regulate multiple aspects of animal reproduction. Most studies investigating the Dmrt gene family were conducted in model organisms from bilateral species, with a particular emphasis on gene function in male sex determination. It is still unclear whether the E. ancora Dmrt (EaDmrt) genes found in basal metazoans such as cnidarians share similar characteristics with orthologs in other metazoans. In this study, seven full Dmrt gene transcript sequences for a gonochoric coral, Euphyllia ancora (phylum: Cnidaria; class: Anthozoa), were obtained through transcriptome data mining, RT-PCR analysis, rapid amplification of cDNA ends, and sequencing. These EaDmrts were subjected to quantitative assays measuring temporal and tissue-specific expression. Results demonstrated a unique gene expression pattern for EaDmrtE, which is enriched in female germ cells during the spawning season. Based on the phylogenetic analyses performed across the homologous Dmrt genes in metazoans, we found that the female-specific EaDmrtE gene is not related to the DM1 gene of Acropora spp. coral nor to Dmrt1 of vertebrates, which are involved in sexual reproduction, especially in sex determination (vertebrate Dmrt1). Additionally, high levels of EaDmrtE transcripts detected in unfertilized mature eggs are retained in newly formed zygotes but decrease during embryonic development. We suggest that the newly discovered gene may play a role in oogenesis and early embryogenesis as a maternal factor in corals. Therefore, the sexual reproduction-associated Dmrt gene(s) should have arisen in cnidarians and might have evolved multiple times in metazoans.

Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals.

Vitellogenin (Vg) is a major yolk protein precursor in numerous oviparous animals. Numerous studies in bilateral oviparous animals have shown that Vg sequences are conserved across taxa and that Vgs are synthesized by somatic-cell lineages, transported to and accumulated in oocytes, and eventually used for supporting embryogenesis. In nonbilateral animals (Polifera, Cnidaria, and Ctenophora), which are regarded as evolutionarily primitive, although Vg cDNA has been identified in 2 coral species from Cnidaria, relatively little is known about the characteristics of yolk formation in their bodies. To address this issue, we identified and characterized 2 cDNA encoding yolk proteins, Vg and egg protein (Ep), in the stony coral Euphyllia ancora. RT-PCR analysis revealed that expression levels of both Vg and Ep increased in the female colonies as coral approached the spawning season. In addition, high levels of both Vg and Ep transcripts were detected in the putative ovarian tissue, as determined by tissue distribution analysis. Further analyses using mRNA in situ hybridization and immunohistochemistry determined that, within the putative ovarian tissue, these yolk proteins are synthesized in the mesenterial somatic cells but not in oocytes themselves. Furthermore, Vg proteins that accumulated in eggs were most likely consumed during the coral embryonic development, as assessed by immunoblotting. The characteristics of Vg that we identified in corals were somewhat similar to those of Vg in bilaterian oviparous animals, raising the hypothesis that such characteristics were likely present in the oogenesis of some common ancestor prior to divergence of the cnidarian and bilaterian lineages.

Germ cell development in the scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa).

Sexual reproduction of scleractinian coral is among the most important means of establishing coral populations. However, thus far, little is known about the mechanisms underlying coral gametogenesis. To better understand coral germ cell development, we performed a histological analysis of gametogenesis in Euphyllia ancora and characterized the coral homolog of the Drosophila germline marker gene vasa. The histological analysis revealed that E. ancora gametogenesis occurs in the mesenterial mesoglea between the mesenterial filaments and the retractor muscle bands. The development of germ cells takes approximately one year in females and half a year in males. Staining of tissue sections with an antibody against E. ancora Vasa (Eavas) revealed anti-Eavas immunoreactivity in the oogonia, early oocyte, and developing oocyte, but only faint or undetectable reactivity in developing oocytes that were >150 µm in diameters. In males, Eavas could be detected in the spermatogonia and primary spermatocytes but was only faintly detectable in the secondary spermatocytes, spermatids, and sperms. Furthermore, a reverse transcription-polymerase chain reaction analysis and Western blotting analysis of unfertilized mature eggs proved the presence of Eavas transcripts and proteins, suggesting that Eavas may be a maternal factor. Vasa may represent a germ cell marker for corals, and would allow us to distinguish germ cells from somatic cells in coral bodies that have no distinct organs.