Coral reefs are threatened globally by the rising seawater temperatures associated with climate change (Hoegh-Guldberg et al., 2017) yet some coral populations have demonstrated a remarkable degree of thermal resilience [e.g., corals from thermally variable reefs: Barshis et al. (2013) and Safaie et al. (2018); but see also Klepac and Barshis (2020)]. Plasticity in coral response to elevated temperatures has led to the exploration of techniques targeting the active enhancement of coral thermal tolerance (e.g., assisted evolution; van Oppen et al., 2015). Since initial inquiry into the capacity of transgenerational acclimation in corals (Putnam and Gates, 2015), several studies have examined the effects of adult conditioning (temperature and/or pCO₂) on offspring performance (e.g., Bellworthy et al., 2019; Putnam et al., 2020). However, the potentially diverse proteomic responses across coral life stages and between coral hosts and their dinoflagellate endosymbionts remain uncharacterized. We consequently sought to explore the proteomic effects of thermal conditioning using colonies of the brooding coral Pocillopora acuta that had been exposed to either control (26°C) or experimentally elevated temperatures (29.5°C) for three reproductive cycles. We specifically employed a quantitative proteomics approach known as “isobaric tags for relative and absolute quantification” (iTRAQ; SCIEX) to assess cellular strategies for responding to high temperature by directly targeting the molecules that enact physiological changes in coral and Symbiodiniaceae cells.

Of the 601 proteins that passed QC (mean sequencing coverage ± SD = 12.3 ± 11.3%; Supplementary Table 1 and Supplementary Material), 540 (90%) were from the host, with only 38 (6%) from the endosymbiotic dinoflagellates (Figure 1A). The origin of the remaining 23 proteins (4%) could not confidently assigned. This ∼540:38 (∼14:1) host:endosymbiont ratio is significantly higher (X² test, p < 0.0001) than the ∼2:1 adult biomass ratio of this coral (deduced by Mayfield et al., 2014) and could reflect an annotation bias associated with the fact that the nucleic acid databases queried were not derived from the same samples whose proteomes were analyzed herein; this idea is explored in greater detail in the Supplementary Material.

When looking at the 74 DCPs obtained across all comparisons (∼12% of the proteome; Supplementary Table 2), the compartmental breakdown was also skewed in favor of the host (76% of DCPs); only 12 dinoflagellate DCPs were uncovered (16% of DCPs) (Figure 1B). When compared to all sequenced proteins, this represents an enrichment of endosymbiont DCPs (6% of all proteins vs. 16% of DCPs; Fisher’s exact test, p < 0.01). Furthermore, 10% and 32% of the host and dinoflagellate proteins, respectively, were differentially concentrated across samples (X²₁ = 987, p < 0.001). When considering only the temperature-responsive proteins, six of 540 host coral proteins (1%) and three of 38 endosymbiont proteins (8%) were affected by temperature. This statistically significant (X²₁ = 5.9, p = 0.01) compartmental difference suggests that the dinoflagellate proteome is more thermo-responsive than that of their coral hosts. The same result was observed in previous mRNA- (Mayfield et al., 2014) and protein- (Mayfield et al., 2018) based works with adult specimens of this coral species.

Of the 74 DCPs, 58 and five were affected by life stage and temperature, respectively (with 11 affected by both; Figure 1C). Ten of the 16 temperature-responsive proteins (Table 1) were documented only in the larvae; only three proteins were affected by temperature in adults and not in larvae (with three affected by high-temperature exposure in both) (Figure 1D). Only one protein (de novo.id25751.tr59102) was up-regulated at high temperatures in both adults and their larvae, though the identity of this 376-amino acid (AA) protein could not be resolved using traditional bioinformatics approaches. Given the fact that it was the only protein of more than 600 sequenced that increased significantly in concentration at high temperatures in both adult corals and their offspring, identifying it should be a priority for future research. This protein could perhaps serve as a biomarker for sub-lethal temperature stress.

A Symbiodiniaceae chloroplast ATP synthase and a host large subunit ribosomal protein (L35e) were both down-regulated at high temperatures in both life stages (Table 1). The former is directly responsible for establishing ATP levels in the chloroplast, and its down-regulation at elevated temperatures may signify that chloroplast metabolism was impacted. In plants, expression is essentially completely halted in the dark such that ATP hydrolysis ceases; this is presumably due to the need to conserve ATP (Kohzuma et al., 2017). This down-regulation of chloroplast ATP synthase might therefore represent an ATP conservation strategy occurring at sub-lethal, but still potentially stress-inducing temperatures.

Different cellular pathways were affected by life stage (Figures 2A,B) and temperature (Figures 2C,D) in coral hosts and their dinoflagellate endosymbionts. Indeed, no protein was differentially concentrated across temperatures in both P. acuta and Symbiodiniaceae. Photosynthesis and chloroplast-related processes were the functions most likely to be affected by host life stage (Figure 2B) and high-temperature exposure (Figure 2D and Table 1) in the dinoflagellates. Others have found Symbiodiniaceae photosynthesis proteins to undergo changes in concentration in response to elevated temperature exposure (Takahashi et al., 2008; Hoogenboom et al., 2012). In the coral hosts, protein trafficking and translation were the pathways most impacted by high-temperature exposure (Figure 2C), as also documented in thermally challenged sea anemones (Oakley et al., 2017).

Whereas only adult corals were manipulated in earlier projects, we had the opportunity to compare the proteomic responses of adult corals and their larval offspring in this study. Not only did the larval proteomes change more dramatically upon high-temperature exposure than those of their parents, but different proteins were affected as well (Figures 3A–D); only three of the 16 temperature-responsive proteins showed a congruent response between life stages (Table 1). Furthermore, the aforementioned high-temperature induction of proteins involved in protein trafficking was mainly attributed to the larval response alone (Figures 3B,D). Collectively, the observations that the protein-level responses to sub-lethal, high-temperature exposure differ between (1) host corals and their endosymbionts and (2) adult and larval corals highlight the need to consider both the holobiont response and life stage when assessing coral resistance and resilience in a warming ocean. Given the low level of replication in our study, we recommend a future analysis of additional samples to more rigorously elucidate the effects of temperature on the meta-proteome of this model coral-dinoflagellate endosymbiosis.

All data within this manuscript can be found on the University of California San Diego’s “MassIVE” repository (accession #MSV000087874), as well as the Proteome Xchange repository (accession #PXD027523).

CJM, TYF, WBH, and IMC conceived the experiment. CJM performed the experiment. ABM conducted the lab work. ABM and CJM undertook the data analysis. ABM, CJM, and IMC wrote the manuscript in consultation with TYF and WBH. All authors contributed constructive feedback throughout the experiment and manuscript preparation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.