NCCOS Assessment: Use of Omics to Support Coral Interventions: Proteomics of gametes collected from Acropora cervicornis, Acropora palmata, Orbicella faveolata to Optimize Sexua...

This dataset is comprised of proteomics results to better understand gamete compatibility factors. The dataset includes gametes from three species, Acropora cervicornis, Acropora Palmata, Orbicella faveolate, which were collected in three locations near Key Largo, Florida (Ofav: Horseshoe Reef, Apal: North Dry Rocks, Acer: Carysfort) during three different time periods (August 2022, August 2023, and September 2023).

Successful coral sexual reproduction is foundational to the achieving coral reef restoration, yet reproductive failure is a significant bottleneck to realizing restoration goals. The overall goal of this project is to identify factors that are involved in gamete compatibility and build on this knowledge to improve fertilization success and other downstream choke points in reproduction, such as larval settlement. The 2023 project was an extension of our previous work in genotype compatibility and kinship for Acropora cervicornis (Acer), Acropora palmata (Apal), and Orbicella faveolata (Ofav). The goals of this portion of the overall project were 1) in collaboration with the Drake Lab at the Medical University of South Carolina, we sought to identify glycans present in egg and sperm gametes across multiple genotypes and within three different coral species using Matrix Assisted Laser Desorption/Ionization (MALDI) imaging spectrometry, and 2) correlate the glycan expression with fertilization rates between egg and sperm gametes of the same species.

Methods:

Egg and sperm gametes from three different coral species were collected from reefs around Key Largo during spawning season in August 2022 and August/September 2023. Fertilization trials were conducted within 3 hours after gamete collection. To our knowledge, this glycan imaging method has never before been used with coral gametes therefore, a significant amount of method development and trial and error was needed, which is defined in the specific methods sections.

Fertilization trials: Fertilization trials were conducted by performing reciprocal crosses. Gamete bundles from a single coral genet were collected in a 15 mL tube and incubated at room temperature until bundles lyse. Sperm were transferred to a clean 15 mL tube without disrupting ova. Sperm were diluted and concentration was calculated using a cellometer. Sperm were diluted and added to the appropriate vials. Ova were washed with artificial sea water and added to the appropriate vials. The gametes were incubated together for 4-6 hours prior to fixing for counting.

Gamete preservation. After gametes were combined for fertilization assays, the remaining sperm and ova were preserved for other molecular analyses. For glycan imaging, sperm and ova were fixed and stored at 4C until processing.

Proteomic Analysis: Gametes from collected bundles were frozen at -20C in protein lysis buffer (5% SDS with protease inhibitor). The proteins were extracted by tip sonicating on ice and then centrifuged at 2,000 rcf for 5 minutes. The supernatant was transferred to a new tube and then centrifuged at 10,000 rcf for 10 minutes. The supernatant was transferred to a new tube and 50 ul was reserved for protein quantification. Protein was quantified using Pierce BCA Protein Assay Kit.

Based on quantification, a volume equivalent to 100 ug was diluted to a volume of 80 ul in 5% SDS. Samples were reduced with 10 ul DTT at 60*C for 10 minutes. After cooling to room temperature (10 minutes), samples were alkylated with calcium acetamide and incubating in dark for 30 minutes. Alkylation was quenched by adding 12% phosphoric acid. Protein samples were digested following Protofi's S-trap protocol, using a 1:20 trypsin:sample ratio and incubating for 2 hours at 47C. The resulting peptides were evaporated until dry and resuspended in 100 ul 0.1% formic acid in water. Peptide concentration was quantified using Pierce Quantitative Fluorometric Peptide Assay kit.

Peptides in solution were sent to University of Arkansas and analyzed using LC-MS/MS analysis. Peptide and proteins were identified using Mascot running against a known Orbicella faveolata annotated metagenome. Resulting search data was imported to Scaffold and identifications were accepted at <1% local FDR for peptides and proteins.

Normalized spectral abundance factor was derived from normalized spectral counts and intensity based on absolute quantification (iBAQ) intensity data.

Hollings Marine Laboratory

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Project accession: PXD057823

Token: DJjiY09bxcWh

Project Name: Proteomics of gametes collected from Acropora cervicornis, Acropora palmata, Orbicella faveolata

Project accession: PXD057823

Token: DJjiY09bxcWh

Project DOI: Not applicable

Will become publicly available after publication of a peer-reviewed manuscript.

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Sampling Instrument: Orbitrap Fusion Lumos LC-MS/MS

Software: MaxQuant

Gametes from three species of coral (Orbicella faveolata, Acropora cervicornis,and Acropora palmata) were collected across two spawning years (2022, 2023). Fertilization rates of reciprocal crosses (performed in triplicate) were compared with proteomics of individual gametes.

Proteomic Analysis

Peptides digested from isolated proteins were identified from annotated genomes

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These data were generated after collecting gametes spawned from Acropora palmata, Acorpora cervicornis, and Orbicella faveolata off the coast of Key Largo in August 2022 and August/September 2023.