A mass mortality of the tropical echinoderm Diadema antillarum (urn:lsid:marinespecies.org:taxname:124332) occurred beginning in January 2022 and continuing to at least July 2022. We sought to identify flaviviruses associated with this condition by performing viral metagenomics and tissue transcriptomics. We prepared these libraries from grossly normal, abnormal and reference specimens collected from St Thomas, U.S. Virgin Islands (USVI), and Saba, Caribbean Netherlands. This dataset includes the accession information for the sequences performed and archived at the National Center for Biotechnology Information (NCBI) Sequence Read Archive. While we were unsuccessful in recovering flaviviruses from tissues of these organisms, we were able to detect additional pathogenic agents (Philaster apodigitiformis; Ciliophora) from transcriptomes. 

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Collection

Specimens of Diadema antillarum (urn:lsid:marinespecies.org:taxname:124332)  were collected by snorkel and transported to the lab in seawater buckets. The specimens were photographed, and vivisected into tissue components (body wall, spine, gonad, digestive tract and coelomic fluid), which were then frozen in liquid N2 for transport to the laboratory at Cornell University. 

Transcriptomic Sequencing 

Transcriptomes from coelomic fluid of grossly normal (n = 2) and abnormal (n = 2) individuals from an affected site and of grossly normal individuals from a reference site (n = 2) were prepared alongside grossly normal (n = 2) aboral body wall tissues from a reference site to examine expressed genes and microorganisms associated with infection. RNA was extracted from coelomic fluid (300 μl) and body wall tissues (200 mg) using the Zymo Quick-RNA Tissue/Insect Kit using on-column deoxyribonuclease I digestion per the manufacturer’s protocols. Transcriptome libraries were prepared from purified RNA extracts using the Zymo RiboFree kit and sequenced on a single run of Illumina MiSeq using a Nextera DNA Library Prep Kit at the Biotechnology Resource Center at Cornell University. All sequences have been deposited at National Center for Biotechnology Information (NCBI) Sequence Read Archive under Bioproject PRJNA900371 and SRA accession numbers SRR22260770 to SRR22260777.

Viral Metagenomic Sequencing

Small subsamples of each specimen were prepared for viral metagenomic sequencing broadly following the approach of Ng et al. (2011). Briefly, tissues were homogenized in sterile, 0.02 µm filtered phosphate buffered saline by beating using Zymo ZR Basher Beads (part number S6012-50) in a Biospec Instruments homogenizer for 1 min at maximum speed. The homogenates were centrifuged at 3,000 × g for 1 min to remove large cell debris, before filtering the supernatant through 0.2 µm pore size polyethersulfone syringe filters. Filtered homogenates were treated with 5U Turbo DNase (Invitrogen; part number AM2238), 5U Benzonase nuclease (Sigma Aldrich; part number E1014) and 20U RNase ONE (Promega; part number M4261) for 2 hours at 37 °C to digest extracellular and non-ribosome-bound nucleic acids. Following incubation, RNA was extracted from subsamples of purified virus-sized material using the Zymo Viral RNA kit (part number R1034) before storing RNA at −80 °C until further processing.

RNA in viral extracts was amplified using the SeqPlex RNA kit (Sigma Aldrich; part number SEQXE-10RXN, n = 11 libraries). Subsequent specimens were amplified using the TransPlex kit (WTA2; Sigma Aldrich; part number WTA2-10RXN, n = 3 libraries), which was used in prior RNA viral metagenomic work. Amplification products were purified using the Zymo DNA Clean and Concentrator -5 (part number D4004), quantified by Pico Green fluorescence (Invitrogen; part number P11496), and submitted for sequencing at the Cornell Biotechnology Resource Center. Libraries were prepared for sequencing using the Nextera Flex kit (Illumina; part number 20018704) and sequenced on the Illumina MiSeq platform using the 500 bp Nano kit. Sequences from all libraries were archived at NCBI under Bioproject PRJNA1117494 and SRA accessions SRR29258987 to SRR29258999.

- Imported "Virome_Transcriptome_Metadata.txt" into the BCO-DMO system
- Converted "Date" to ISO 8601 YYYY-MM-DD format
- Removed the N and W indicators, in accordance with BCO-DMO style for the "Latitude" and "Longitude", making the W values negative
- Renamed NCBI identifier fields to include more standard and descriptive titles
- Exported file as "985619_v1_virome_transcriptome.csv"

Scientific names in the data were checked using World Register of Marine Species (WoRMS) Taxon Match. All scientific names in the data are valid and accepted names as of 2025-10-01.

Coverage: Northeastern Pacific Ocean

NSF Award Abstract:

Marine diseases pose considerable risks to invertebrates, such as sea cucumbers, in the face of changing ocean conditions. While many invertebrate diseases are driven by pathogens, the interplay between animal biology and environmental conditions often mediates the outcome of the pathogen-host relationship. Sea cucumbers are ecologically and economically important animals that occur in a wide range of marine habitats. This project aims to decipher how the interaction between the biology of sea cucumbers, environmental conditions, and a newly-discovered type of virus, seemingly innocuous under typical conditions, may lead to lethal disease in giant Pacific sea cucumbers in the U.S. West Coast. The study includes surveys in coastal regions in southeast Alaska, Washington, and California as well as laboratory experiments manipulating seawater oxygen concentrations, temperature, and simulated microalgal blooms. The project engages community scientists, fishers, high school students, and indigenous groups, and supports training of one graduate and several undergraduate students. A workshop that brings together scientists across marine ecology, disease, and veterinary disciplines is planned to prepare a handbook of best practices in marine disease investigation.

Metagenomic and community-level sequencing efforts have revealed an astonishing diversity of viruses associated with grossly normal marine invertebrates. The vast majority of detected viruses likely represents asymptomatic infections under typical conditions but may generate pathology in hosts under changing environmental conditions. This project investigates the ecology of a group of enveloped positive sense single-stranded RNA viruses (flaviviruses) that this research team has recently discovered in the giant California sea cucumber Apostichopus californicus by addressing three hypotheses: 1) Aquatic insect-only Flaviviruses (aiFVs) do not cause gross pathology under typical conditions; 2) aiFVs proliferate and generate clinical and gross pathology under suboxic stress; and 3) Periodic increases in primary production and mean temperature excursions cause aiFV proliferation and subsequently exacerbate holothurian disease process. The study comprises a restricted survey of aiFV diversity via amplicon sequencing and their prevalence within and between populations, development of an antibody-based approach for aiFV detection, and examination of aiFV behavior in concert with host transcription and veterinary pathology. The study includes field surveys and in laboratory manipulative experiments.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.