DOI: 10.5061/dryad.02v6wwq9z
title: Trade-off between photo-symbiosis and innate immunity influences cnidarian’s response to pathogenic bacteria

description: Mutualistic relationships with photosynthetic organisms are common in
 cnidarians, which form an intracellular symbiosis with dinoflagellates in
 the family Symbiodiniaceae. The establishment and maintenance of these
 symbionts are associated with the suppression of key host immune factors.
 Due to this, there are potential trade-offs between the nutrition
 cnidarian hosts gain from their symbionts and their ability to
 successfully defend themselves from pathogens. To investigate this
 potential trade-off, we utilized the facultatively symbiotic polyps of the
 upside-down jellyfish Cassiopea xamachana and exposed aposymbiotic and
 symbiotic polyps to the pathogen Serratia marcescens. Symbiotic polyps had
 a lower probability of survival following S. marcescens exposure.
 Gene expression analyses 24 hours following pathogen exposure indicate
 that symbiotic animals mounted a more damaging immune response, with
 higher levels of inflammation and oxidative stress likely resulting in
 more severe disruptions to cellular homeostasis. Underlying this more
 damaging immune response may be differences in constitutive and
 pathogen-induced expression of immune transcription factors between
 aposymbiotic and symbiotic polyps rather than broadscale immune
 suppression during symbiosis. Our findings indicate that in facultatively
 symbiotic polyps, hosting symbionts limits C. xamachana’s ability to
 survive pathogen exposure, indicating a trade-off between symbiosis and
 immunity that has potential implications for coral disease research.
description: # Trade-off between photo-symbiosis and innate immunity influences
 cnidarian’s response to pathogenic bacteria
 [https://doi.org/10.5061/dryad.02v6wwq9z](https://doi.org/10.5061/dryad.02v6wwq9z) The study associated with this code tested differences between survival, acidic organelle activity, and gene expression between symbiotic and aposymbiotic *Cassiopea xamachana* in response to *Serratia marcescens*. ## Description of the data and file structure ### Physiology/survival data ##### microscope\_images- directory with all raw microscopy images, used to measure mean eGFP (symbiont density) or RFP (acidic organelle activity) fluorescence, contains the following directories:  * Lysotracker_24hrs- directory of images of polyps treated with lysotracker to stain for acidic organelle activity following 24 hours of exposure to their respective treatment groups. The following abbreviations are used in the naming scheme: AB- aposymbiotic polyp exposed to *S. marcensens*, AC- aposymbiotic polyp control, SB- symbiotic polyp exposed to *S. marcensens*, symbiotic polyp control  * Lysotracker_72hrs- directory of images of polyps treated with lysotracker to stain for acidic organelle activity following 72 hours of exposure to their respective treatment groups. The following abbreviations are used in the naming scheme: AB- aposymbiotic polyp exposed to *S. marcensens*, AC- aposymbiotic polyp control, SB- symbiotic polyp exposed to *S. marcensens*, symbiotic polyp control  * Symbiont_density- directory of images of polyps under eGFP to measure symbiont density and confirm the significant reduction of symbionts in aposymbiotic polyps. The following abbreviations are used: apo- aposymbiotic polyp, symbio- symbiotic polyp ##### phys\_code\_inputs - directory with input files for phys\_code.Rmd, includes README and CSV files of survival data, symbiont density, and acidic organelle activity data, contains the following: 1. Apo_conf_10-26.csv is a file with the mean eGFP fluorescence of aposymbiotic and symbiotic polyps. Treatment abbreviations: Symb- symbiotic polyp, Apo: aposymbiotic polyp. 2. SM_kap_meyer.csv is a file with the right censored survival data for Kaplan Myer survival analysis. Only pathogen-exposed polyps are included in this data as no control polyps died. The treatment column 1 represents symbiotic polyps and 2 represents aposymbiotic polyps. Fustat indicates if the animal lived past the end of the experiment (0) or died (1). 3. lyso_final_for_R.csv is a file with the mean RFP fluorescence of polyps at 24 and 72 hours following treatment exposure and lysotracker staining. Treatment abbreviations: C= control, SM= *S. marcensens*, status abbreviations: S= symbiotic, A= aposymbiotic.  ### Gene expression data #### Command line inputs raw RNASeq reads- published on NCBI SRA: BioProject ID PRJNA1077944 *C. xamachana* filtered cds gene models from the Ohdera et al. draft genome: https://mycocosm.jgi.doe.gov/Casxa1/Casxa1.home.html #### R analyses inputs ##### Inputs that can be generated from public data/code in this repository: quants_casxa - Salmon output directory, contains quant.sf files for each sample, primary input for tximport casxa_counts.csv - count data matrix, output from tximport Casxa_GO.tsv- tab-separated file of gene ID and its associated GO terms, can be made using Casxa1_annoation_eggnog_edit_down.csv ##### SM\_gene\_expression\_analysis\_inputs - directory with input files for SM\_gene\_expression\_analysis\_R.Rmd contains the following: ###### Inputs for gene expression analyses * casxa_txt2gene.csv - text to gene file for tximport * cas_SM_samples.csv - sample list for tximport * Cas_SM_metadata.csv - metadata for DESeq, abbreviations: SM= S. marcensens exposed, apo= aposymbiotic, symbio= symbiotic.  * Casxa1_annoation_eggnog_edit_down.csv - eggNOG-mapper annotation of gene models, unused annotation columns removed * casxa_PRR_blast.csv - list of Cassiopea pattern recognition receptors previously identified in Emery et al. 2021 * casxa_STRINGID.csv - conversion file for gene model IDs compatible with the STRING annotation - casxa_geneinfo_mod150_STRING.csv - WGCNA output, includes module membership and STRING ID of contigs ###### Inputs for data visualization  * heat_by_category_expression_order.csv - input for Figure 4 (DEG heatmap) * control_GO_figure_input.csv - GO enrichment inputs for Figure 5 control symbiotic state comparison * SM_GO_figure_input.csv - GO enrichments inputs for Figure 5 S marc symbiotic state comparison * apo_GO_figure_input.csv - GO enrichments inputs for Figure 5 aposymbiotic pathogen response comparison * symbio_GO_figure_input.csv - GO enrichments inputs for Figure 5 symbiotic pathogen response comparison * black_10_enrich.csv - STRING enrichments for the black module selected for visualization in Figure 6 * green_10_enrich.csv - STRING enrichments for the green module selected for visualization in Figure 6 * grey_10_enrich.csv - STRING enrichments for the gey60 module selected for visualization in Figure 6 * magenta_10_enrich.csv - STRING enrichments for the magenta module selected for visualization in Figure 6 * red_10_enrich.csv - STRING enrichments for the red module selected for visualization in Figure 6 * Turq_10_enrich.csv - STRING enrichments for the Turquoise module selected for visualization in Figure 6 ## Sharing/Access information Raw reads are available on NCBI SRA: BioProject ID PRJNA1077944 ## Code/Software phys_code.Rmd- R code used for survival analysis, analyses for differences in symbiont density, and analyses for differences in acidic organelle activity command_line.Rmd- Linux code to trim raw RNAseq reads and quantify transcripts, software versions: Fastp version 0.23.3, Salmon version 1.9.0, BBsplit version 39.01. SM_gene_expression_analysis_R.Rmd- R code used to import Salmon data, call differentially expressed genes, perform Weighted Gene Co-expression Network Analysis, and visualize gene expression results  All R code runs with R version 4.2.2. ## Supplemental information supplemental files can be found in this repository and as electronic supplementary material published with the manuscript  **file descriptions** Supplementary File 1- RNAseq read quality and mapping rates Supplementary File 2- PC1 GO enrichments Supplementary File 3- tables of all DEGs Supplementary File 4- tables of all GO enrichments from differential expression analysis Supplementary File 5- all WGCNA module STRING enrichments