Continuation of Microscopic and Microbial Insights into the Stony Coral Tissue Loss Disease (SCTLD) Outbreak Across Multiple Coral Species on Florida's Coral Reef

EXECUTIVE SUMMARY
 

Four integrated research tasks were performed using molecular biology, histology, and immunohistochemistry to provide insights into a possible mechanism of microbial infection that results in tissue loss, as well as potential pathogen(s) associated with stony coral tissue loss disease (SCTLD). For Task 1, molecular tools were used to characterize the endolithic communities of fungi, bacteria, and archaea from apparently healthy coral colonies, and affected and unaffected portions of diseased colonies sampled in 2016 and 2017. For Task 2, histological methods were used to characterize the endosymbiotic dinoflagellates in tissue from apparently healthy colonies and affected and unaffected portions of diseased colonies collected from Florida reefs or obtained following laboratory experiments conducted by Dr. Andrew Baker’s research team at the University of Miami to examine their role in the pathogenesis of SCTLD. For Task 3, immunohistochemistry was used to investigate the role of programmed cell death (PCD) vs. necrosis in SCTLD. For Task 4, histoslides of apparently healthy and SCTLD-affected coral tissue samples were digitized using an Aperio ScanScope slide scanner. The resulting virtual microscopy files were uploaded with pertinent metadata on each sample to Florida’s Fish and Wildlife Research Institute cloud storage. These virtual slides will be used for further study and interpretation by histopathologists considering the results of all SCTLD research.
 

Results from Task 1 indicated that the combination of proteinase K and lysozyme incubations, with an additional DNA cleanup step, did not increase PCR amplification efficiency of MCAV samples for bacterial/archaeal analysis as desired. However, these steps did increase the number of samples that amplified for fungal analyses. Preliminary analysis indicated that the endolithic coral microbiome (bacteria/archaea and fungi) varied in community composition based on host coral species and location, but not necessarily health state (with the caveat that diseased samples were limited in size for this analysis and additional analyses need to be performed to resolve fungal taxonomic identification). Additional bioinformatic analyses will be conducted to further investigate specific microbes driving observed differences and to examine the functional potential of the coral endolithic microbiome during different disease states. Results from Task 2 showed widespread necrosis and degradation within both SCTLD-exposed ‘healthy’ and ‘lesion’ samples. Lytic necrosis was seen in 45% of all histoslides examined (n=22), representing all five species of coral observed and all species of Symbiodinium (excluding Cladocopium). These results are consistent with what was seen in the 2016 FWRI samples in which zooxanthellae were often degraded or lysing in the apparently healthy, diseased, and unaffected tissue samples from all four species of coral. Lytic necrosis was observed in all species, with few to no zooxanthellae in these lesions that were usually restricted to the gastrodermis, and the mesoglea and epidermis remained intact. Gastrodermis adjacent to these lesions included pale-staining algal cells with vacuolation and swelling, then cell wall disintegration and lysing (ghosting). Results from Task 3 revealed that staining indicative of apoptosis was consistently observed in tissues of SCTLD-affected Pseudodiploria strigosa, Montastraea cavernosa, and Orbicella faveolata, indicating that this mode of cell death may be involved in SCTLD pathology. However, apoptosis was also observed in samples without an apparent lesion, indicating that apoptosis may be an early indicator of disease before degradation can be detected morphologically, or may be normally expressed to some degree for routine epithelial cell replacement. In all species, staining for apoptosis was significantly more prevalent in diseased tissues, indicating that this process is increasingly expressed in disease, even if it is also part of healthy tissue functioning to a lesser degree. Histoslides were selected from 8 coral species examined by FWRI and scanned to capture the tissue section details digitally for Task 4. The image files have been uploaded and linked through a spreadsheet by filename to enable viewing of the slides from the Florida Fish and Wildlife Conservation Commission’s website on SCTLD using Aperio’s ImageScope software.
 

The results of this project contributed to a better understanding of disease mechanisms and insights on potential pathogenic agents involved in the progression of SCTLD in reef-building corals. Additional studies using these tools are needed to answer questions about the etiology and impact of SCTLD on Caribbean reefs.