Li-Hsueh Wang

Effects of Gonadal Steroids on Brain Serotonergic and Aromatase Activity During the Critical Period of Sexual Differentiation in Tilapia, Oreochromis mossambicus

The effects of gonadal steroids on brain serotonin (5‐HT) and aromatase activity during the critical period of sexual differentiation were investigated in tilapia, Oreochromis mossambicus. Treatment of tilapia with 17β‐oestradiol (E2) between days 7 and 10 posthatching resulted in a significant increase in the female : male ratio as determined at day 90, and a significant reduction in brain 5‐HT content. Treatment between days 10 and 20, or between days 20 and 30, had no significant effect. Since the 5‐HT system may influence sexual differentiation, we examined the effects of para‐chlorophenylalanine (p‐CPA), a 5‐HT synthesis inhibitor. As with E2, treatment of tilapia with p‐CPA between days 7 and 10 posthatching resulted in a significant increase in the female : male ratio. Again, treatment between days 10 and 20, or between days 20 and 30, had no significant effect. Both p‐CPA and E2 significantly depressed brain aromatase activity when administrated between days 7 and 10, but not subsequently. In tilapia treated between days 7 and 10, the brain 5‐HT content was lowered by E2 to an extent similar to that seen with p‐CPA, which is consistent with the suggestion that the effect of E2 on sexual differentiation may be mediated by the 5‐HT system. Treatment of tilapia with 17α‐methyltestosterone (MT), by contrast, resulted in a reduction in the female : male ratio, and treatment was most effective when given between days 10 and 20. The period of maximal effect of MT upon sex ratio appears to coincide with the ability of MT to induce an increase in brain aromatase activity.

Proteomic analysis of symbiosome membranes in Cnidaria–dinoflagellate endosymbiosis

Symbiosomes are specific intracellular membrane‐bound vacuoles containing microalgae in a mutualistic Cnidaria (host)–dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin‐XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X‐100 soluble and insoluble fractions, were subjected to 2‐D SDS‐PAGE and identified by MS using an LC‐nano‐ESI‐MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti‐apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.

Assessing the Impacts of Experimentally Elevated Temperature on the Biological Composition and Molecular Chaperone Gene Expression of a Reef Coral

Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment.

Ratiometric imaging of gastrodermal lipid bodies in coral–dinoflagellate endosymbiosis

Cnidaria–dinoflagellate endosymbiosis is the phenomenon of autotrophic symbionts living inside the gastrodermal cells of their animal hosts. The molecular mechanism that regulates this association remains unclear. Using quantitative microscopy, we now provide evidence that the dynamic lipid changes in gastrodermal “lipid bodies” (LBs) reflect the symbiotic status of the host cell and its symbiont in the hermatypic coral Euphyllia glabrescens. By dual-emission ratiometric imaging with a solvatochromic fluorescent probe, Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one), we showed that the in situ distribution of polar versus neutral lipids in LBs in living gastrodermal cells and symbionts can be analyzed. The ratio of Nile red fluorescence at red (R) versus green (G) wavelength region (i.e., R/G ratio) correlated with the relative molar ratio of polar (P) versus neutral (NP) lipids (i.e., P/NP ratio). The R/G ratio in host LBs increased after bleaching, indicating a decrease in neutral lipid accumulation in gastrodermal cells. On the other hand, neutral lipid accumulation inside the symbiont LBs resulted in gradual decreases of the R/G ratio as a result of bleaching. In comparison with the bleaching event, there was no relative lipid concentration change in host LBs under continual light or dark treatments as shown by insignificant R/G ratio shift. Patterns of R/G ratio shift in symbiont LBs were also different between corals undergoing bleaching and continual light/dark treatment. In the latter, there was little lipid accumulation in symbionts, with no resulting R/G ratio decrease. These results, demonstrating that the symbiotic status positively correlated with morphological and compositional changes of lipid bodies, not only highlight the pivotal role of LBs, but also implicate an involvement of lipid trafficking in regulating the endosymbiosis.

Lipid Content and Composition during the Oocyte Development of Two Gorgonian Coral Species in Relation to Low Temperature Preservation

Our previous studies have suggested that chilling sensitivity of coral oocytes may relate to their relatively high lipid intracellular content and lipid composition. The distribution of lipids during the oocyte development was determined here for the first time in two gorgonian species (Junceella juncea and Junceella fragilis). The main lipid classes in the two gorgonian oocytes were total lipid, wax ester, triacylglycerol, total fatty acid, phosphatidylethanolamine and phosphatidylcholine. The results indicated that early stage oocytes of J. juncea and J. fragilis were found to have increased lipid content than late stage oocytes. The content of wax ester was significantly higher in the early stage oocytes of two gorgonian corals (51.0±2.5 and 41.7±2.9 µg/mm3/oocyte) than those of late stage oocytes (24.0±1.4 and 30.4±1.2 µg/mm3/oocyte, respectively). A substantial amount of phosphatidylethanolamine and total fatty acid was detected at each stage of oocyte development in two gorgonian ranges from 107 to 42 µg/mm3/oocyte and 106 to 48 µg/mm3/oocyte, whilst low levels of phosphatidylcholine were found in two gorgonian oocytes. The levels of total lipid in the late stage oocytes of J. juncea were significantly higher than those of J. fragilis. The observed differences may partially be related to different habitat preferences as higher lipid levels in J. juncea, a deeper-water coral species exposed to lower temperature seawater, might relate to adjustments of cell membranes in order to increase membrane fluidity.

Fatty acid and phospholipid syntheses are prerequisites for the cell cycle of Symbiodinium and their endosymbiosis within sea anemones.

Lipids are a source of metabolic energy, as well as essential components of cellular membranes. Although they have been shown to be key players in the regulation of cell proliferation in various eukaryotes, including microalgae, their role in the cell cycle of cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses remains to be elucidated. The present study examined the effects of a lipid synthesis inhibitor, cerulenin, on the cell cycle of both cultured Symbiodinium (clade B) and those engaged in an endosymbiotic association with the sea anemone Aiptasia pulchella. In the former, cerulenin exposure was found to inhibit free fatty acid (FFA) synthesis, as it does in other organisms. Additionally, while it also significantly inhibited the synthesis of phosphatidylethanolamine (PE), it did not affect the production of sterol ester (SE) or phosphatidylcholine (PC). Interestingly, cerulenin also significantly retarded cell division by arresting the cell cycles at the G0/G1 phase. Cerulenin-treated Symbiodinium were found to be taken up by anemone hosts at a significantly depressed quantity in comparison with control Symbiodinium. Furthermore, the uptake of cerulenin-treated Symbiodinium in host tentacles occurred much more slowly than in untreated controls. These results indicate that FFA and PE may play critical roles in the recognition, proliferation, and ultimately the success of endosymbiosis with anemones.

Norterpenoids and Related Peroxides from the Formosan Marine Sponge Negombata corticata

Six norterpenes including negombatoperoxides A and B (4 and 5), the inseparable epimers negombatoperoxides C and D (6 and 7), negombatodiol (8), and negombatolactone (9), in combination with three known compounds, (+)-nuapapuin B (1), (+)-nuapapuin B methyl ester (2), and (+)-aikupikoxide C (3), were isolated from the Formosan marine sponge Negombata corticata. In addition, 6,6-dimethylundecane-2,5,10-trione (10) was isolated for the first time from a natural source. Their structures, including relative configurations, were elucidated on the basis of interpretation of spectroscopic data and by the application of the empirical rule established by Capon and MacLeod. The absolute configurations of 8 and 9 were established by the application of Moshers method and comparison of CD data with known lactones, respectively. Cytotoxicity of these isolates against human breast carcinoma, human liver carcinoma, and human lung carcinoma cell lines was evaluated.

Photoperiod affects the development of central neurotransmitter systems of tilapia, Oreochromis mossambicus

The effects of photoperiod on the development of central neurotransmitters were investigated with tilapia, Oreochromis mossambicus. Zero-day-old (the hatching day) tilapia were raised in three different photoperiods (light/dark cycle): 12/1, 24/0, and 0/24 h, respectively. On the 5th day, brain serotonin (5-HT), norepinephrine (NE), gamma-aminobutyric acid (GABA), and glutamate (Glu) contents were quantified by a high-performance liquid chromatograph with electrochemical detection. Similar experiments were performed on the 5-, 10-, 15-, 20-, and 25-day-olds. These results showed that the photoperiod influenced both brain NE and GABA contents during its respective restricted period, before days 10 posthatching. Brain 5-HT content was influenced, either facilitated or suppressed according to the developing stage, whereas, brain Glu content was not altered by the different photoperiod exposure throughout the present studies.

Diversity in skeletal architecture influences biological heterogeneity and Symbiodinium habitat in corals

Scleractinian corals vary in response to rapid shifts in the marine environment and changes in reef community structure post-disturbance reveal a clear relationship between coral performance and morphology. With exceptions, massive corals are thought to be more tolerant and branching corals more vulnerable to changing environmental conditions, notably thermal stress. The typical responses of massive and branching coral taxa, respectively, are well documented; however, the biological and functional characteristics that underpin this variation are not well understood. We address this gap by comparing multiple biological attributes that are correlated with skeletal architecture in two perforate (having porous skeletal matrices with intercalating tissues) and two imperforate coral species (Montipora aequituberculata, Porites lobata, Pocillopora damicornis, and Seriatopora hystrix) representing three morphotypes. Our results reveal inherent biological heterogeneity among corals and the potential for perforate skeletons to create complex, three-dimensional internal habitats that impact the dynamics of the symbiosis. Patterns of tissue thickness are correlated with the concentration of symbionts within narrow regions of tissue in imperforate corals versus broad distribution throughout the larger tissue area in perforate corals. Attributes of the perforate and environmentally tolerant P. lobata were notable, with tissues ∼5 times thicker than in the sensitive, imperforate species P. damicornis and S. hystrix. Additionally, P. lobata had the lowest baseline levels of superoxide and Symbiodinium that provisioned high levels of energy. Given our observations, we hypothesize that the complexity of the visually obscured internal environment has an impact on host-symbiont dynamics and ultimately on survival, warranting further scientific investigation.

Lipid Content and Composition of Oocytes from Five Coral Species: Potential Implications for Future Cryopreservation Efforts

Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.