Dataset: 16S rRNA sequences for outplanted Acropora cervicornis sexual recruits

Name: 16S rRNA sequences for outplanted Acropora cervicornis sexual recruits collected from Mote Marine Laboratory and Looe Key National Marine Sanctuary from 2020-2022
Published: 2024-08-18
Keywords: outplant, horizontal transmission, oceans

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Cite This Dataset

Spatial Extent: N:24.56257 E:-81.40009 S:24.54069 W:-81.43652

Mote's in situ nursery. 24.56257, -81.40009, Looe Key National Marine Sanctuary 24.54093, -81.43539 and 24.54069, -81.43652

Temporal Extent: 2020-10-07 - 2022-04-21

Project:

Collaborative Research: Tracking the interacting roles of the environment, host genotype, and a novel Rickettsiales in coral disease susceptibility (Coral Rickettsiales)

Principal Investigator:

Rebecca Vega Thurber (Oregon State University, OSU)

Co-Principal Investigator:

Erinn M. Muller (Mote Marine Laboratory, Mote)

Scientist:

Grace J. Klinges (Mote Marine Laboratory, Mote)

BCO-DMO Data Manager:

Lynne M. Merchant (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-08-18

Restricted:

Validated:

Current State:

Preliminary and in progress

16S rRNA sequences for outplanted Acropora cervicornis sexual recruits collected from Mote Marine Laboratory and Looe Key National Marine Sanctuary from 2020-2022

Abstract:

A pervasive association exists between the bacterium Aquarickettsia rohweri and Caribbean Acropora, as A. rohweri dominates microbiomes of field-collected samples of this coral species. In particular, this bacterial species is highly abundant in genotypes of Acropora cervicornis susceptible to white band disease with reduced abundance in disease-resistant genotypes. A. rohweri, a member of the order Rickettsiales, is hypothesized to be an obligate symbiont dependent on the coral holobiont for nutrition and energy. Many other closely-related parasites within Rickettsiales are transmitted vertically, and A. rohweri is unlikely to persist in a free-living stage due to its limited metabolic capabilities. This bacterial parasite was therefore expected to be transmitted vertically between host generations. However, phylogenomic analyses of Acropora spp. and A. rohweri did not reveal the co-evolutionary characteristics expected of a vertically transmitted symbiont. These characteristics could be obscured, however, by horizontal transmission between hosts. The identification of A. rohweri in evolutionarily distant aquatic hosts ranging from ctenophores to sponges also strongly supports horizontal transmission of this species. To better understand the transmission dynamics of Aquarickettsia, populations of this bacteria were quantified in early life stages of A. cervicornis (gametes, planula larvae, early sexual recruits, and year-old juveniles) produced and raised in the land-based nursery at Mote Marine Laboratory in the Florida Keys. These corals were produced via controlled two-parent crosses involving six different genotypes across three annual spawning events. We found that Aquarickettsia was absent from captive-raised individuals though present in parental genotypes maintained in Mote Marine Laboratory’s in situ coral nursery. In March 2021, offspring were transferred to the same in situ nursery or outplanted to reef plots either near to or far from (> 50 m) adult A. cervicornis to determine if proximity to other colonies affected parasite acquisition. Corals were sampled one week, one month, and two months post-transplantation to assess timing of Aquarickettsia infection and to examine shifts in the coral microbiome overall due to transplantation. We determined that proximity to wild or outplanted A. cervicornis influenced Aquarickettsia acquisition in outplanted conspecifics that lacked Aquarickettsia prior to outplanting. Only corals that were in close proximity to adult A. cervicornis acquired Aquarickettsia, and acquisition took between 1 and 6 months. Importantly, corals outplanted far from previously-extant outplants did not acquire Aquarickettsia by a full year after transplantation to the reef. This suggests that horizontal transmission is important in the acquisition of this putative parasite, and the parasite is likely transmitted by other A. cervicornis or reef organisms primarily associated with outplanted A. cervicornis (e.g. corallivorous snails, ciliates, bearded fireworms). We also observed that not all conspecifics from the same families acquired the parasite, even within the same site. This suggests that Aquarickettsia acquisition may require prolonged contact with a vector, or that there is an element of chance in exposure to and subsequent infection with Aquarickettsia.

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Methods & Sampling

To sample each coral, 6-8 polyps were excised from outplanted corals using bone cutters and temporarily stored in individual Whirl-Paks on ice until reaching shore. Upon shore, samples were transferred from Whirl Parks using flame-sterilized tweezers placed in a 1.5mL microcentrifuge tube containing 1mL of DNA/RNA shield (Zymo Research, R1100-250, Irvine, CA, USA). Samples were transferred to a -80℃ freezer for long-term storage. In preparation for DNA extractions, the samples were removed from the -80℃ freezer and thawed on ice. With flame-sterilized tweezers, half of the biomass was transferred to a Disruptor Tube (Omega Bio-Tek, Norcross, GA, USA), the other half was kept as a bioarchive and returned to -80℃. DNA from each sample was isolated utilizing the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) with slight modifications to the manufacturer’s protocol to increase yield. DNA isolates were stored at -80℃. DNA quantity and quality was assessed utilizing a NanoDrop spectrophotometer (Thermo Fisher Scientific™, Waltham, MA, USA). Samples were submitted to MR DNA for 16S rRNA PCR amplification and sequencing (www.mrdnalab.com, Shallowater, TX, USA). Amplification of the 16S rRNA gene was conducted using the 515F-806R primer set, which targets the V4 region of the 16S rRNA, with barcodes on the forward primer (Apprill et al., 2015). The 16S rRNA gene V4 variable region was amplified via a 30-cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD) under the following conditions: 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 10 minutes was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Samples were multiplexed using unique dual indices and were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads (Beckman Coutler, CA, USA). Then the pooled and purified PCR product was used to prepare an Illumina DNA library. A PCR negative control was included in library preparation but did not produce a viable library. Paired-end sequencing was performed at MR DNA on an Illumina MiSeq following the manufacturer's guidelines.

Sampling locations:

Mote's in situ nursery. Approximate central coordinate 24.56257, -81.40009.
Site in Looe Key National Marine Sanctuary: U-10, U-11, and U-12. Approximate central coordinate 24.54093, -81.43539.
Site in Looe Key National Marine Sanctuary: Unnamed site west of U-11. Approximate central coordinate 24.54069, -81.43652.

Data Processing Description

No processing was performed on raw reads after sequencing before submission to NCBI.

BCO-DMO Processing

Five files were joined together on unique columns. Four files were submitted and one file was downloaded with NCBI with extra metadata related to the 4 submitted files.

This is the process to create the final dataset.

1) In the file Accessions_outplant_16S.tsv, columns related to the files rather than the data were removed. Given the accession numbers, the user will be able to find and download relevant files. The columns about the instrument and not the data were removed since instrument metadata is noted in the instruments section on the dataset page.

Columns removed: object_status, filetype, filename, filename2, filename3, filename4, assembly, and fasta_file. Along with platform, and instrument model.

2) In the file outplant_MIMARKS.survey.host-associated.6.0.xlsx, two empty columns named sample_title and bioproject_accession were removed.

Columns were added giving the term equivalent of *env_broad_scale and *env_local_scale notations.

Columns removed: sample_title and bioproject_accession

3) In the file SRA_metadata_outplant.xlsx, columns related to the files rather than the data were removed. Given the accession numbers, the user will be able to find and download relevant files. Columns about the instrument and not the data were removed since instrument metadata is noted on the dataset page.

Columns removed: filetype, filename, filename2, filename3, filename4, assembly, and fasta_file. Along with platform, and instrument model.

4) The data manager downloaded a file from NCBI using their SRA Run Selector for the BioProject. The file contains experiment SRX accession numbers to include into the final data file. The file is called SraRunInfo_from_ncbi.csv.

It contains the columns Run, Experiment, LibraryName, LibraryStrategy, LibrarySelection, LibrarySource, LibraryLayout, SRAStudy, BioProject, Sample, BioSample, TaxID, ScientificName, SampleName, Submission.

The following columns that are NCBI specific columns and a taxonomic id were removed.

Columns removed: TaxID, LibraryStrategy, LibrarySelection, LibrarySource, and LibraryLayout

5) To start the joins of all the files, the file SraRunInfo_from_ncbi.csv was joined with SraRunInfo_from_ncbi.csv on the BioSample accession number.

Duplicate columns were removed.

The resulting joined table was named join_1.

6) For the second join, table join_1 was joined with the file SRA_metadata_outplant.xlsx on the column sample_name.

Duplicate columns along with NCBI specific columns library_strategy, library_source, library_selection, and library_layout were removed.

The resulting joined table was named join_2.

7) For the third join, table join_2 was joined with the file outplant_MIMARKS.survey.host-associated.6.0.xlsx on the column sample_name.

The duplicate column sample_name was removed.

The resulting joined table was named join_3.

8) For the fourth join, table join_3 was joined with the file metadata_outplant_with_ELS.txt on the column library_id and sample_id.

Duplicate columns were removed. The columns *sample_name, new_id, and sample_id have some variance in naming so they are not exact duplicate columns and were kept in the table.

The resulting joined table was named 924594_v1_rrna_seq_outplanted_acropora_cervicornis_sexual_recruits because it will be the final file name.

9) Field names were renamed to BCO-DMO naming conventions. Asterix were removed and periods were replaced with underscores.

10) The combined lat and lon field with direction was converted to individual columns lat and lon without direction. The lon column was converted to a negative value because the lon direction is West.

11) The collection_date was converted from the format day-Month abbreviation-year where the day is not padded and the year is a two digit year to the format of %Y-%m-%d where the day is padded and the year is a 4 digit year.

12) Columns were removed that that were reformatted such as the lat_lon column and the original collection_date column.

13) The metadata in the table was reordered to be at the start of the dataset and the data at the end of the dataset.

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1923926
NSF Division of Ocean Sciences	OCE-1923836

Deployments

None available yet

Instruments

Automated DNA Sequencer

Supplied Name:

Supplied Description:

Illumina MiSeq

Instrument Type

Generic Name: Automated DNA Sequencer

Acronym: Automated Sequencer

Generic Description:

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

bone cutter

Supplied Name:

Supplied Description:

Instrument Type

Generic Name: bone cutter

Generic Description:

A bone cutter is a surgical instrument used to cut bones or coral fragments.

Thermo Scientific NanoDrop spectrophotometer

Supplied Name: NanoDrop spectrophotometer

Supplied Description:

Instrument Type

Generic Name: Thermo Scientific NanoDrop spectrophotometer

Acronym: NanoDrop spectrophotometer

Generic Description:

Thermo Scientific NanoDrop spectrophotometers provide microvolume quantification and purity assessments of DNA, RNA, and protein samples. NanoDrop spectrophotometers work on the principle of ultraviolet-visible spectrum (UV-Vis) absorbance. The range consists of the NanoDrop One/OneC UV-Vis Spectrophotometers, NanoDrop Eight UV-Vis Spectrophotometer and NanoDrop Lite Plus UV Spectrophotometer.

Parameters

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Dataset: 16S rRNA sequences for outplanted Acropora cervicornis sexual recruits