Additional file 2 of Gill-associated bacteria are homogeneously selected in amphibious mangrove crabs to sustain host intertidal adaptation

Additional file 2: Method S1. Quantification of ammonia concentration in crab gills. Male individuals of Cranuca inversa and Thalamita crenata were collected from the Ibn Sina Field Research Station mangrove at KAUST (KSA) and kept in dedicated aquaria with fresh sediment for C. inversa and filtered fresh seawater flushed with air to maintain an oxygen saturation of 98%, at 21°C, 1 atm (assessed through a Fibox4 logger, Presence, Regensburg, Germany). After 12 h of acclimation, 10 individuals of each species were sacrificed, and the left gills were extracted. Gills were weighed and soaked with 300 µL of sterile ultrapure water (Invitrogen, Waltham, USA). Samples were then manually homogenised with plastic pestles for 1.5 µL tubes and centrifuged for 5 min, 13000g. After centrifugation, the supernatants were collected to be centrifuged again for another 10 min at 13000g. The final supernatant was used to quantify ammonia concentration in the crab gills using the ammonia assay kit MAK310 (Merck, Darmstadt, Germany) following the manufacturer's instructions. Fluorescence readings were performed with a TECAN infinite 200 pro spectrophotometer (TECAN, Grödig, Austria) in 96-well clear bottom black polystyrene microplates (Corning, NY, USA). Results were calculated following manufacturers’ indications and normalised on the fresh weight of initial gill tissue. Table S1. Pairwise comparison of the bacterial beta-diversity among Sites and Species (including sediments). Table S2. List of FISH probes used in this study. Table S3. General statistics for metagenomes and assemblies of fiddler gill and burrow sediments microbiomes. Table S4. Summary of 16S rRNA gene sequences retrieved from individual metagenomes under study. Table S5. List of KEGG orthology (KO) further investigated in this study related to carbon, sulfur, and nitrogen metabolism as well as the detoxification of sulfur compounds and xenobiotics. Table S6. General information and statistics for metatranscriptomes and assemblies from the gill tissues of the fiddler crab C. inversa collected in the Red Sea, KAUST coastline mangroves. Figure S1. Rarefaction (A) and Goods’ coverage index (B) of the bacterial 16S rRNA gene amplicon sequencing dataset. Figure S2. Eulero-Venn Diagram that shows the shared OTUs (numbers represent the percentage weighted for the OTUs relative abundance) among sediment, seawater and crab gills, and Taxonomy of the overall samples (A) and considering only the samples from Red Sea (B). Figure S3. (A) Taxonomical composition of bacterial communities in T. crenata and C. inversa highlights the large presence of Ilumatobacter sp. in the semiterrestrial fiddler crab gills and its paucity in the aquatic crabs. Notably, the “other Actinobacteria” detected in T. crenata mainly belonged to Propionibacteriaceae and Microtrichaceae. (B) Quantification of ammonia concentration in the crab gills. Significantly different ammonia concentrations on the gill of the swimming crabs Thalamita crenata and the fiddler crabs Cranuca inversa (Mann-Whitney test, U=14, p<0.0052, n=10). Figure S4. Constrained analysis of principal coordinates of the phylogenetic distances (alpha PD) among the bacterial microbiomes across (A) sites and (B) crab species. Figure S5. Scanning electron microscope imaging of fiddler crabs gill studied where we can see the constant coverage of the bacterial layer in all the species investigated. Figure S6. Bright-field (a) and FISH negative controls (b) of fiddler crabs gill lamellae. Figure S7. Metagenomic protein-coding gene sequence space of crab and bulk sediment microbiomes. (a) Total counts of predicted protein-coding genes in individual samples. (b) Relative abundance of prokaryotic (bacteria and archaea) relative to eukaryotic genes in sequenced metagenomes. (c) Phylum-level taxonomic breakdown of bacterial genes indicates. Actinobacteria’s prevalence in crab samples. Figure S8. Abundance and taxonomic assignment of unamplified 16S rRNA gene sequences retrieved from crab and bulk sediment metagenomes. (a) Total counts of 16S rRNA genes in individual samples. (b) Relative abundance of prokaryotic (bacteria and archaea) 16S rRNA genes in sequenced metagenomes. (c) Phylum-level taxonomic breakdown of bacterial 16S rRNA genes indicates Actinobacteria’s prevalence in crab samples. Figure S9. Krona graph showing the taxonomic breakdown of the representative protein-coding gene catalogue predicted from all metagenomes. Figure S10. Total counts of predicted protein-coding genes and the corresponding domain. Additional information is provided in Table S5.