Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Histopathology of Growth Anomaly Affecting the Coral, Montipora capitata: Implications on Biological Functions and Population Viability

Growth anomalies (GAs) affect the coral, Montipora capitata, at Wai'ōpae, southeast Hawai'i Island. Our histopathological analysis of this disease revealed that the GA tissue undergoes changes which compromise anatomical machinery for biological functions such as defense, feeding, digestion, and reproduction. GA tissue exhibited significant reductions in density of ova (66.1–93.7%), symbiotic dinoflagellates (38.8–67.5%), mesenterial filaments (11.2–29.0%), and nematocytes (28.8–46.0%). Hyperplasia of the basal body wall but no abnormal levels of necrosis and algal or fungal invasion was found in GA tissue. Skeletal density along the basal body wall was significantly reduced in GAs compared to healthy or unaffected sections. The reductions in density of the above histological features in GA tissue were collated with disease severity data to quantify the impact of this disease at the colony and population level. Resulting calculations showed this disease reduces the fecundity of M. capitata colonies at Wai'ōpae by 0.7–49.6%, depending on GA severity, and the overall population fecundity by 2.41±0.29%. In sum, GA in this M. capitata population reduces the coral's critical biological functions and increases susceptibility to erosion, clearly defining itself as a disease and an ecological threat

Patterns of Coral Disease across the Hawaiian Archipelago: Relating Disease to Environment

In Hawaii, coral reefs occur across a gradient of biological (host abundance), climatic (sea surface temperature anomalies) and anthropogenic conditions from the human-impacted reefs of the main Hawaiian Islands (MHI) to the pristine reefs of the northwestern Hawaiian Islands (NWHI). Coral disease surveys were conducted at 142 sites from across the Archipelago and disease patterns examined. Twelve diseases were recorded from three coral genera (Porites, Montipora, Acropora) with Porites having the highest prevalence. Porites growth anomalies (PorGAs) were significantly more prevalent within and indicative of reefs in the MHI and Porites trematodiasis (PorTrm) was significantly more prevalent within and indicative of reefs in the NWHI. Porites tissue loss syndrome (PorTLS) was also important in driving regional differences but that relationship was less clear. These results highlight the importance of understanding disease ecology when interpreting patterns of disease occurrence. PorTrm is caused by a parasitic flatworm that utilizes multiple hosts during its life cycle (fish, mollusk and coral). All three hosts must be present for the disease to occur and higher host abundance leads to higher disease prevalence. Thus, a high prevalence of PorTrm on Hawaiian reefs would be an indicator of a healthy coral reef ecosystem. In contrast, the high occurrence of PorGAs within the MHI suggests that PorGAs are related, directly or indirectly, to some environmental co-factor associated with increased human population sizes. Focusing on the three indicator diseases (PorGAs, PorTrm, PorTLS) we used statistical modeling to examine the underlying associations between disease prevalence and 14 different predictor variables (biotic and abiotic). All three diseases showed positive associations with host abundance and negative associations with thermal stress. The association with human population density differed among disease states with PorGAs showing a positive and PorTrm showing a negative association, but no significant explanatory power was offered for PorTLS

In vitro culture of cells derived from larvae of the staghorn coral Acropora millepora

Previous attempts to culture cells from corals or\ud other cnidarians have been unsuccessful. These efforts\ud have, however, generally made use of adult tissue as\ud starting material. Early developmental stages are potentially more appropriate for the initiation of cell cultures, as the expectation is that a greater proportion of the cell population is undifferentiated and may have the intrinsic ability of unlimited cell renewal. To explore this idea, cell cultures were initiated from five key stages of coral development, and the presence of coral cells monitored by polymerase chain reaction (PCR) using coral-specific primers. After 4 weeks, semi-quantitative PCR implied that coral cells were better represented in cultures initiated from planulae than in those derived from earlier developmental stages. Coral cells were detected in cultures initiated from planulae for up to 10 weeks, but after this time, extensive contamination by the protist Thraustochytrium sp. was observed

Comprehensive characterization of skeletal tissue growth anomalies of the finger coral Porites compressa

The scleractinian finger coral Porites compressa has been documented to develop raised growth anomalies of unknown origin, commonly referred to as "tumors". These skeletal tissue anomalies (STAs) are circumscribed nodule-like areas of enlarged skeleton and tissue with fewer polyps and zooxanthellae than adjacent tissue. A field survey of the STA prevalence in Oahu, Kaneohe Bay, Hawaii, was complemented by laboratory analysis to reveal biochemical, histological and skeletal differences between anomalous and reference tissue. MutY, Hsp90a1, GRP75 and metallothionein, proteins known to be up-regulated in hyperplastic tissues, were over expressed in the STAs compared to adjacent normal-appearing and reference tissues. Histological analysis was further accompanied by elemental and micro-structural analyses of skeleton. Anomalous skeleton was of similar aragonite composition to adjacent skeleton but more porous as evidenced by an increased rate of vertical extension without thickening. Polyp structure was retained throughout the lesion, but abnormal polyps were hypertrophied, with increased mass of aboral tissue lining the skeleton, and thickened areas of skeletogenic calicoblastic epithelium along the basal floor. The latter were highly metabolically active and infiltrated with chromophore cells. These observations qualify the STAs as hyperplasia and are the first report in poritid corals of chromophore infiltration processes in active calicoblastic epithelium areas. © 2006 Springer-Verlag

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)

Abstract Background Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians. Methods Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. Results We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. Conclusions In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies