Ariana S. Huffmyer, Kevin H. Wong, Danielle M. Becker-Polinski, Emma Strand, Tali Mass, Hollie M. Putnam
This repository contains data, scripts, and analysis for early life history symbiotic and metabolic responses in the vertically-transmitting reef building coral Montipora capitata across developmental stages.
Figure 1. (A) Variables measured across Montipora capitata development. Filled squares indicate response variable was sampled at the respective time point; black squares indicate the response was not measured or data is not available. Color corresponds to major life history grouping (eggs=brown, embryos=yellow, larvae=cyan, metamorphosed polyps=green, attached recruits=pink). Hpf indicates hours-post-fertilization. (B) Photographs of 1 - Montipora capitata colony; 2 - Egg-sperm bundles fertilized in 50 mL falcon tubes; 3 - Conical larval rearing chambers; 4 - Planula larvae, note pigmentation due to presence of symbiont cells; 5 - Attached recruits on settlement plug.
In this project, we collected physiology, metabolic, metabolomic, transcriptomic, and microbiome data across egg, embryo, larvae, metamorphosed polyp, and attached recruit lifestages, shown in Figure 1. This work was conducted in 2020 at the Hawaii Institute of Marine Biology and the Coral Resilience Lab.
Developmental stages measured included:
- Eggs: Fertilized eggs from pooled fertilization of egg and sperm from wildtype egg-sperm bundles collected in Kaneohe Bay, Oahu, Hawaii.
- Embryos: Embryos developing starting at cleavage through to the gastrula stage.
- Larvae: Larvae sampled after development into the swimming planula stage.
- Metamorphosed polyps: Polyps that underwent metamorphoses, prior to attachment to the substrate. Collected free floating in the water or resting on the bottom of rearing containers.
- Attached recruits: Metamorphosed and attached recruits attached to aragonite plug substrate.
The respository is organized in folders for Data, Scripts, Output, and Figures. The Data folder contains raw data for each analysis. The Output folder contains any data produced by analyses or output through analytical pipelines. The Scripts folder contains all R scripts and .md files that contain any bioinformatics pipelines used. The Figures folder contains all figures and visualizations produced from analyses.
For all analyses, consistent lifestage and developmental time point identifiers are imported and matched to the Data/lifestage_metadata.csv file. Note that in each dataset there are variations in the nomenclature used for each developmental time point. Final figures are all standardized to the correct metadata names that identify lifestages as "Lifestage (hours post-fertilization)".
If multiple .Rmd files are used for the same data type, the scripts are numbered for the order in which the analysis is run.
Physiological data was collected for larval size (calculated as larval volume) and symbiont densities. Larval size is available for eggs through metamorphosed polyps. Symbiont density is calculated as cells per individual in eggs through metamorphosed polyps and as cells per unit surface area for attached recruits. Physiology data is analyzed using the Physiology_Analysis.Rmd script.
Respirometry data was collected in late embryos, larvae, and metamorphosed polyps. Metabolic rates measured included:
- Light-enhanced respiration: Respiration rates calculated as nmol oxygen consumed per individual per minute measured in the dark following measurement of photosynthesis rates in the light.
- Net and gross photosynthesis: Net photosynthesis rates were calculated as nmol oxygen produced per individual per minute with gross photosynthesis calculated as net photosynthesis + oxygen consumed through respiration.
- Gross photosynthesis to respiration ratios: The ratio of P to R was calculated by dividing gross photosynthesis by respiration rates. A P:R >1 indicates higher oxygen production than consumption.
Respirometry data is analyzed by first extracting metabolic rates using the LoLinR package in the 1_Respirometry_Extraction.Rmd script and then plotted and analyzed in the 2_Respirometry_Plotting.Rmd script.
Gene expression was characterized across life stages with TagSeq sequencing at the University of Texas Austin. Sequences were trimmed and underwent QC followed by alignment to the latest (version 3) Montipora capitata genome available through Rutgers University. Sequences were aligned and a gene count matrix was calculated using bioinformatics pipelines detailed in the TagSeq_BioInf_genomeV3.md file in the Scripts/TagSeq/Genome_V3/ folder.
The gene count matrix was then imported into R with multivariate visualizations and WGCNA analysis in the 1_WGCNA_Mcap_V3.Rmd script. Results of WGCNA were then functionally annotated using the reference genome with accompanying functional annotation. Functional annotation of genes was conducted using GO-Seq in the 2_WGCNA_GoSeq_V3.Rmd script. Expression of genes with specific GO terms were examined with the 3_Gene_Expression_Indicators_V3.Rmd.
Sequences were also aligned to the symbiont genome (Cladocopium sp. and Durusdinium sp.) for exploratory purposes. This data is not included in the manuscript due to low symbiont sequences and alignment. Scripts for this exploratory analysis are found in the Symbiont folder in the Scripts/TagSeq folder.
Raw sequence files can be found on NCBI SRA under BioProject PRJNA900235.
Metabolomics was characterized across development with untargetted metabolomic analysis conducted at Rutgers University Metabolomics Shared Resource. Ion count tables of identified metabolites first underwent QC and filtering with multivariate visualization and analysis in the 1_metabolomics.Rmd script. Metabolomic profiles across lifestages were then analyzed using a WCNA approach in the 2_metabolomics_WGCNA.Rmd script. Specific metabolites were examined in the 3_metabolite_indicators.Rmd script.
Raw metabolomics files can be found on the Open Science Framework project.
Symbiont ITS2 sequences were analyzed using the SymPortal pipeline. Symbiont communities at the DIV level were analyzed in the ITS2_strains.Rmd file. Filtering and subsampling were conducted in this R script.
Raw sequence files can be found on NCBI SRA under BioProject PRJNA900235.
Bacterial 16S sequences were analyzed using the Mothur MiSeq SOP pipeline detailed in the Mothur_bioinformatics.md file. Bacterial communities were analyzed in the 16S_Mothur.Rmd file. Due to low DNA extraction and bacterial sequence abundance, samples from the earliest lifestages are not included in analysis.
Raw sequence files can be found on NCBI SRA under BioProject PRJNA900235.
For each data type above, relative change in metrics of interest was calculated across development. These relative change indicators were then plotted together in an integrative plot in the Integration_Relative_Change.Rmd script.
Any questions about this project and the contents of this repository can be directed to Ariana S. Huffmyer at ashuffmyer (at) uri.edu.