Location: Emerging Pests and Pathogens Research
Project Number: 8062-22410-007-047-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Nov 1, 2023
End Date: Jul 2, 2024
Objective:
Florida’s Coral Reef is currently experiencing a multi-year disease-related mortality event, that has resulted in massive die-offs in multiple coral species. Approximately 21 species of coral, including both Endangered Species Act-listed and the primary reef-building species, have displayed tissue loss lesions which often result in whole colony mortality. First observed near Virginia Key in late 2014, the disease has since spread to the northernmost extent of Florida’s Coral Reef, and southwest past the Marquesas in the Lower Florida Keys. The best available information indicates that the disease outbreak is continuing to spread west and throughout the Caribbean.
A recent study had identified viral-like particles (VLPs) within coral tissue that were associated with the microalgae symbionts, which appeared to resemble filamentous RNA viruses (Work et al., 2021). The authors of that study suggested they belonged to the viral family Flexiviridae based on morphology alone, but they did not have supporting sequencing data. Also, these VLPs were observed in both diseased and apparently healthy corals, so no connection between these VLPs and disease has yet to be established. There have been other described viruses associated with corals and their symbionts unrelated to Stony coral tissue loss disease (SCTLD) (Wilson et al., 2001; Lohr et al., 2007; Thurber et al., 2009). Viruses typically outnumber bacteria in a 10:1 ratio in marine systems, so their mere presence does not provide strong evidence for their role as a primary pathogen.
This project is building upon the current work of the ARS research group. The objective is to identify the viruses and microbes associated with diseased corals, determining if they are specific to SCTLD lesions, and establishing if there are any associations between potential pathogens and the start of SCTLD lesions through time. This is a multi-phase project with Phase I focusing on samples collection and the beginning of samples processing. Phase II will focus on samples analysis. This award is for Phase I.
Approach:
Laboratory pipeline to characterize virus populations in coral samples. The number of samples to be analyzed and sampling scheme will scale with the budget requested and be guided by the results of the transmission assay. Samples will be prepared by cryogenic lysis and separated for DNA, RNA and protein extractions. Purified DNA will be sent to the Meyer Lab at the University of Florida for microbiome analysis (Task 6). Total RNA will be extracted from coral samples using the SpectrumTM Plant Total RNA kit (Sigma-Aldrich) as per the manufacturers' instructions including a DNase digestion step. Equimolar amounts of RNA samples from the transmission assay experiments, or that originated from samples displaying virus-like symptoms will be pooled into composite samples containing up to 10 RNA individual samples. To enrich for virus transcripts, ribosomal RNA will be depleted from the composite samples using QIAseq FastSelect–rRNA Plant Kit (Qiagen). Libraries of cDNA will be prepared from each composite ribodepleted RNA sample using the TruSeq Stranded mRNA kit (Illumina). Libraries will be sequenced in the Genomics Research and Technology Hub at the University of California, Irvine using the Illumina NovaSeq 6000 sequencer.
Bioinformatic analyses, and virus presence confirmation. Computational biology analyses will be performed as detailed previously (Olmedo-Velarde et al., 2021).
Genome nucleotide sequence variation of virus isolates and their respective homologs: The new genome sequences generated in this study will be multiple aligned using the CLUSTALW algorithm implemented in MEGA 11.0.9 (Tamura et al., 2021).
Phylogenetic relationships: Phylogenetic relationships will be inferred from the multiple nucleotide sequence alignments detailed above as well as multiple protein sequence alignments performed in MEGA 11.0.9.
Protein extractions to characterize viral proteins in coral samples. Protein samples will be extracted from the same samples as RNA to facilitate multi-omics data integration. Samples will be prepared for mass spectrometry using trypsin digestion, a technique that is routine for the ARS Ithaca Laboratory (Kruse et al., 2018).
Mass Spectrometry-based proteomics. Mass spectrometry-based proteomics will be carried out at the University of Washington under a USDA ARS Research Support Agreement with the University of Washington.
Mass spectrometry data analysis. Mass spectrometry proteome data will be submitted to ProteomeXchange for access to the community and analyzed using our in house pipeline.
Multi-omics integration. Multi-omics integration promises to reveal novel factors contributing to SCTLD that would not be obvious from the analysis of individual datasets. ARS will use an unsupervised computational method, Multi Omics Factor Analysis (MOFA)(Argelaguet et al., 2018), to examine the sources of biological variation in the omics datasets.
