Kruger, A. J. | Search bioRxiv

bioRxiv2026

Improving coral oxidative stress assessments through compartment-specific lipid peroxidation measurements and increased methodological standardization

Lipid peroxidation (LPO) is widely used as a biomarker of oxidative stress in coral bleaching research, yet its measurement remains poorly standardized across the field. A systematic review of the coral LPO literature reveals substantial variation in methodological approaches, including tissue fraction analysis, lysis protocols, assay choice, and normalization metrics, confounding cross-study comparison and obscuring the biological interpretation of results. We experimentally investigate two key sources of variation: the use of bulk holobiont vs separated host and algal symbiont fractions, and the choice of normalization metric. To do so, we used Montastraea cavernosa (n = 6 colonies) exposed to ambient (28 {degrees}C), heat stress (30.5 {degrees}C), and heat stress + artificial upwelling (AU; heat stress intermitted by daily pulses of cooler water, 30.5/27.5 {degrees}C) conditions in a controlled mesocosm experiment. Using a TBARS-based MDA assay with a lysis buffer optimized for coral tissue, we measured LPO separately in coral host and algal symbiont fractions across four time points throughout the day. Host MDA remained stable across all treatments and time points, consistent with either sufficient antioxidant buffering capacity or thermal acclimation over the experimental period. Algal symbiont MDA, in contrast, exhibited pronounced diel and treatment-specific dynamics, and the two fractions responses were decoupled from one another. Normalizing MDA to coral surface area instead of total protein content produced largely consistent diel and treatment patterns, but the two metrics diverged at specific time points, indicating that normalization choice is not interchangeable and can itself affect interpretation. Together, our literature review and empirical results demonstrate that host and algal symbiont LPO dynamics are not comparable when aggregated and argue for host-symbiont fraction separation and consistent, explicitly reported normalization as minimum standards for interpretable and cross-comparable coral LPO measurement.

biochemistry↗

bioRxiv2025

Deep conservation of mitochondrial HSP60 structure with lineage-specific and context-dependent regulation reflects thermal resilience in cnidarians

Heat shock proteins (HSPs) are ubiquitous molecular chaperones that safeguard proteostasis under stress. We first investigated the expression dynamics of the mitochondrial chaperonin HSP60 across diverse cnidarians to understand its stress-responsive regulation. Using immunoblotting, we quantified HSP60 expression in Pocillopora acuta (reef-building coral), Exaiptasia diaphana (sea anemone), and Cassiopea xamachana (upside-down jellyfish). In P. acuta, HSP60 was not detected at the fragment scale under either control or heat stress, whereas isolated cells exhibited transient HSP60 expression during exposure to both control and heated temperatures (+5 {degrees}C above optimum), indicating that HSP60 regulation in this coral is strongly context-dependent and potentially suppressed at the tissue level. In contrast, E. diaphana and C. xamachana showed gradual, and temperature-dependent accumulation of HSP60 over 24 h under heated conditions (+5 {degrees}C above its thermal optimum), however C. xamachana also displayed constitutive basal expression under control conditions. These contrasting profiles highlight clear lineage-specific differences in HSP60 regulation among cnidarians. The consistent antibody cross-reactivity observed across all three species then prompted us to explore the evolutionary basis of this conservation. Phylogenetic analyses of HSP60 sequences confirmed that cnidarian proteins are orthologous to the canonical vertebrate HSP60 (human HSPD1), demonstrating deep structural and evolutionary conservation of this chaperonin across Metazoa. Collectively, these findings reveal that while HSP60 is evolutionarily ancient and conserved, its regulation under thermal stress varies across lineages and physiological context, reflecting complex modulation of mitochondrial proteostasis in early-diverging metazoans. This lineage- and context-dependent regulatory framework provides new insight into how chaperone plasticity contributes to cnidarian thermal tolerance and the differential susceptibility of reef taxa to bleaching under ocean warming. Significance StatementHeat shock protein 60 (HSP60) is a highly conserved mitochondrial chaperonin critical for maintaining protein homeostasis, yet its regulatory dynamics across early-diverging animal lineages are poorly understood. By first comparing the expression responses of three phylogenetically and ecologically distinct cnidarians--the coral Pocillopora acuta, the sea anemone Exaiptasia diaphana, and the upside-down jellyfish Cassiopea xamachana--we uncovered clear lineage-specific differences in HSP60 regulation. P. acuta showed no detectable HSP60 induction in intact tissue, underscoring strong context-dependence that may prevent the deployment of this critical molecular defense mechanism, reflecting its high thermal susceptibility. In contrast, E. diaphana and C. xamachana displayed gradual, temperature-dependent accumulation aligning with their thermal flexibility, with C. xamachana also displaying constitutive basal levels under control condition. The consistent antibody cross-reactivity across all three species then led us to investigate evolutionary conservation, revealing that cnidarian HSP60s are orthologous to the canonical HSP60 (human HSPD1). This demonstrates that HSP60 is deeply conserved from cnidarians to mammals, yet its stress-responsive regulation has diversified across lineages and physiological contexts. This lineage- and context-dependent regulatory framework illuminates how fundamental differences in chaperone control shape cnidarian stress physiology, offering new mechanistic insight into the cellular basis of coral bleaching susceptibility under ocean warming.

biochemistry↗