Comparative Proteomic Profiling of Antioxidant Pathways in Microbial Crustacean Symbioses

Thesis Abstract

In marine ecosystems, microbial symbionts associated with crustaceans influence host oxidative stress responses, yet the comparative proteomic architecture of antioxidant pathways across distinct microbial crustacean symbioses remains underexplored. This study aims to elucidate how antioxidant networks differ among symbiotic communities and to identify core and variable proteomic signatures that modulate host resilience to reactive oxygen species. Specific objectives are to (i) profile global and targeted antioxidant-related proteins in three crustacean hosts with contrasting symbiont assemblages using tandem mass tag (TMT)–based quantitative proteomics, (ii) compare pathway-level abundances of catalase, superoxide dismutase, peroxiredoxins, glutathione metabolism, and thioredoxin systems across symbioses, (iii) evaluate correlations between symbiont diversity metrics (Shannon index, Simpson index) and host antioxidant protein abundance, and (iv) construct a conceptual model linking microbial community structure to proteomic antioxidant responses under experimentally induced oxidative stress. A mixed-methods approach combines quantitative proteomics with ecological and statistical analyses. The population comprises three crustacean species from distinct benthic habitats (eurythermal, eutrophic, and oligotrophic), each harboring characteristic microbial consortia. A total of 45 individual crustaceans (15 per species) will be collected, ensuring equivalent life stage and health status. Tissue samples (gill and hepatopancreas) will be dissected under sterile conditions for proteomic analysis, while 16S rRNA and metagenomic sequencing will characterize symbiont communities. Proteins will be extracted, labeled with TMT reagents, and analyzed by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatic workflows will annotate antioxidant-related proteins, map them to canonical pathways (KEGG, Reactome), and quantify differential expression across hosts. Data on symbiont diversity will be integrated with proteomic outputs using multivariate analyses. Statistical analyses will employ linear mixed-effects models to test whether antioxidant protein abundance significantly differs by host species and symbiont assemblage, controlling for batch effects. ANOVA will assess intergroup differences in specific enzyme classes (e.g., catalases, peroxidases). Correlation analyses (Spearman’s rank) will explore relationships between diversity indices and antioxidant protein levels. A redundancy analysis (RDA) will examine how microbial community structure explains variance in proteomic profiles. Pathway enrichment and network analyses will identify coordinated antioxidant modules, while structural equation modeling (SEM) will test directed causal links from symbiont diversity to proteomic outputs and oxidative stress markers. The theoretical framework integrates the Holobiont Theory and the Stress Response paradigm, positing that host-microbe assemblages function as an integrated unit where microbial composition modulates host antioxidant capacity. Expected findings include (i) distinct antioxidant proteomes associated with each symbiotic community, with higher relative abundance of peroxiredoxins and glutathione metabolism enzymes in hosts hosting more diverse microbial consortia, (ii) a positive correlation between symbiont diversity and overall antioxidant capacity, as indicated by combined catalase and SOD activities, and (iii) coordinated regulation of thioredoxin and glutathione pathways forming a core antioxidant module across symbioses, albeit with asymmetrical emphasis depending on habitat-derived stressors. These results would delineate conserved and variable components of antioxidant defense shaped by microbial partners. The study contributes to knowledge by explicitly linking microbial community structure to host biochemical resilience via proteomic antioxidant pathways, advancing the Holobiont concept in crustacean systems and informing ecophysiological models of oxidative stress tolerance. Practical implications include improved understanding of how symbioses influence crustacean health in changing environments and potential biomarkers (e.g., specific peroxiredoxin isoforms) for monitoring oxidative stress responses. Limitations include potential confounding effects of age or diet, mitigated by controlled sampling, and the challenge of distinguishing host versus symbiont protein origins, addressed by host–symbiont separation protocols and targeted proteomics. Overall, the study will set a framework for cross-species comparisons of proteomic antioxidant strategies in animal–microbial symbioses and guide future experimental manipulations of symbiont communities to enhance host resilience.