Table of Contents

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

Forty-two specimens of Apostichopus californicus (urn:lsid:marinespecies.org:taxname:529363) were collected by SCUBA divers in Thimbleberry Bay (57.031861N, 135.250972W), near Sitka, Alaska on 10 November 2021 and transported together in plastic tubs to the lab at the University of Alaska Southeast (Japonski Island, Sitka). Specimens were immediately weighed, photographed, and placed into individual mesh containers within 7 x 1200 L outdoor mesocosms (6 specimens per mesocosm) filled with seawater from the nearby Sitka Channel. Two mesocosms served as controls (no amendment), 4 mesocosms were subject to daily organic matter (20 µM) amendment, and 1 mesocosm was continuously sparged with N₂ (Airgas, medical grade; the other 6 mesocosms were bubbled continuously with air). Seawater was subject to 50% volume water change daily and specimens were not fed during captivity. Mesocosms were covered while not sampled (i.e., they were light limited). We selected two organic matter substrates (glucose and peptone) based on their ability to stimulate microbial activity in prior work in addition to two common constituents of dissolved organic matter in coastal environments (N-acetylglucosamine and fucose + rhamnose). We monitored dissolved oxygen levels in each mesocosm using continuous submersible HOBO loggers.

Surface swabs of each individual were collected daily by rubbing a Puritan polyester sterile swab over a ~ 1 cm² area of epidermis. Swabs were cut using clean scissors to remove the polyester tip and placed into 2 mL cryovials containing RNALater. Tube feet samples were collected from each specimen daily using disposable plastic forceps, and feet were placed immediately into cryovials containing RNALater. A 5mm biopsy punch of the dorsal body wall was collected from half of the individuals in each mesocosm at 0, 1, 3, and 6 d, which were then preserved in RNALater. All specimens were photographed and weighed daily. All RNALater preserved samples were frozen at -80°C and transported in liquid N₂ to the laboratory at Cornell University. Animal carcasses were frozen at -20°C and transported on blue ice to Cornell.

Surface swabs were collected from 42 animals over the course of 7 d (sampled on t = 0 d, 1 d, 3 d, 5 d, and 7 d) and frozen at -20°C until further processing. DNA was extracted from frozen swabs using Zymo Quick-DNA Fungal/Bacterial kits (Zymo Research) as per the manufacturer’s protocol. Bacterial communities in sample extracts were identified using dual-barcoded PCR (polymerase chain reaction) amplification and sequencing of the V4 region of the 16S rRNA gene. Each 40 µL PCR reaction comprised 1X PCR master mix (One-Taq Quick-Load 2x Master Mix with Standard; New England Biolabs), 0.125 µM of each barcoded primer (515f; 5’-GTG YCA GCM GCC GCG GTA A-3’ and 806r; 5’-GGA CTA CNV GGG TWT CTA AT-3’), and 2 uL of template (swab extract). 16S rRNA amplicons were pooled at equimolar concentrations using SequalPrep Normalization Plate kit (Invitrogen) and sequenced on one lane of Illumina MiSeq (2 x 250 paired end) at the Cornell University Biotechnology Research Center. 16S rRNA amplicon sequences were submitted to NCBI (BioProject accession number PRJNA947521, see Related Datasets).

- Imported "MIMARKS.survey.host-associated.5.0_Sitka_Sea_Cucumber_16S.xlsx" into the BCO-DMO system
- Converted "collection_date" to ISO 8601 format YYYY-MM-DD
- Removed parameters with no values
- Removed special characters from parameter names
- Split "lat_lon" parameter into "Latitude" and "Longitude"
- Replaced lat and lon values with values provided by submitter for collection location
- Renamed "collection_date" to "swab_collection_date"
- Created a new column, "specimen_collection_date" with values of 2021-11-10, the date all specimens were collected
- Filtered out all rows with "host_subject_id" values that included "BLANK", "Blank", or "Basket", upon submitter request
- Added corresponding BioSample identifiers (provided by submitter) to the dataset
- Added corresponding AphiaIDs and LSIDs for relevant species
- Renamed "misc_param" and "host_taxid" to "replicate_ID" and "host_NCBI_taxid"
- Exported file as "984835_v1_swab_sequence_accessions.csv"

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.