{"paper_id":"4c099656-35aa-45cf-8531-0824cbf6e917","body_text":"Reproductive Resilience: Pathways to Gametogenic Success in Montipora capitata after Bleaching | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reproductive Resilience: Pathways to Gametogenic Success in Montipora capitata after Bleaching E Timmins-Schiffman, E Duselis, T Brown, JB Axworthy, CH Backstrom, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4559943/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Thermal bleaching, or the loss of symbiotic algae that provide most energetic resources for the coral host, is an increasing threat to reefs worldwide and is projected to worsen with climate change. While bleaching is a well-recognized threat, the impact on the process of reproduction in bleaching survivors is not well resolved, despite being central to coral resilience. Montipora capitata can survive bleaching while completing a full gametogenic cycle, offering an ideal system to study gametogenic resilience and physiological tradeoffs. We experimentally bleached fragments of M. capitata colonies and followed their gametogenesis and physiological responses for 10 months (six time points). All bleached colonies produced gametes at the same time as controls, suggesting that reproductive processes were energetically prioritized. However, proteomic analysis revealed tradeoffs and delays in activating key physiological processes earlier in gametogenesis in areas such as skeletal growth and reproductive hormone synthesis. Tradeoffs during the gametogenic cycle, likely a direct response to thermal bleaching, resulted in smaller oocytes from bleached colonies, potentially indicating decreased transfer of parental resources to gametes. While gametogenesis is likely to continue in this species, it is unknown how the viability and success of future offspring may be impacted by future bleaching events. Biological sciences/Ecology/Climate change ecology Biological sciences/Molecular biology/Proteomics thermal stress reproduction histology reef-builder proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Reproduction is essential for species propagation and potential evolutionary adaptation, both of which are critical for species survival amid the unprecedented challenges of a rapidly changing climate. Worldwide coral reefs are experiencing increased frequency and severity of conditions related to climate change and human activity, such as increased sea surface temperatures, ocean acidification, sedimentation, and pollution (reviewed in [ 1 ]). Continuation of successful reproduction under these conditions will likely require both extensive physiological plasticity (e.g. [ 2 ]) as well as high genetic diversity (e.g. [ 3 ]) to enable adaptation to novel environments. Despite the importance of reefs to coastal ecosystems and economies (e.g.,[ 4 ]), the underlying physiological acclimatization needed for continued resilient and successful reproduction in corals experiencing environmental stress are not known. Successful reproduction despite experiencing physiological stress may come at a cost to the reproducing adult. If gametogenesis is energetically prioritized in a resource-limited physiological state, such as after thermal bleaching, colony growth [ 5 ] or even survival and susceptibility to disease [ 6 ] may be at risk. This balance between the long-term benefits of reproduction versus the short-term tradeoffs in growth and health may also have a tipping point. In the coral Montipora digitata , a reduction in photosynthesis led to an energetic shift, prioritizing of gametogenesis to a certain extent. Partially shaded colonies allocated a larger percentage of their available energy to gametogenesis and less to colony growth, whereas fully shaded colonies ceased gametogenesis completely [ 7 ]. This suggests that the phenotypic plasticity has limits and beyond these limits short-term colony survival is prioritized over reproduction. Bleaching is a stress response associated with a variety of environmental stressors that occur when a coral colony no longer maintains its symbiotic relationship with algal endosymbionts and appears pale or white in color. In Hawaiʻi, thermal bleaching typically occurs in August or September when seawater temperature peaks [ 8 ], coinciding with the beginning of a new gametogenic cycle for Montipora capitata [ 9 ]. Various aspects of bleaching recovery in M. capitata have been investigated (e.g., [ 10 ], [ 8 ], [ 11 ]), but specific mechanisms facilitating continued gametogenesis post-bleaching have not been investigated. Specifically, the question of how corals overcome the physiological tax of bleaching at essential points in the gametogenic process to realize full gametogenesis has never been addressed. Uncovering the tradeoffs necessary for gametogenesis post-bleaching could give insight into what is required for population persistence in a changing ocean. M. capitata is a simultaneous hermaphrodite with gametogenesis lasting the better part of a year [ 12 ]. Oogenesis begins in late summer/early fall and progresses through a period of rapid oocyte size increase in the spring until spawning in June or July [ 12 ]. Spermatogenesis is a shorter process, lasting four to five months, from April to June/July [ 12 ]. M. capitata has previously shown resilience in its gametogenic process in the face of environmental stress (e.g. [ 13 ]). Its long gametogenic cycle could make M. capitata more vulnerable to disruptive environmental impacts, but may also provide more time for coral recovery and spawning. The latter seems to be the case for M. capitata , as the species has previously demonstrated high phenotypic canalization in gametogenesis in response to a variety of environmental drivers, such as ammonium enrichment [ 14 ], sedimentation [ 12 ], and thermal stress [ 13 ]. Resilience in reproduction is essential to overall population survival in changing environments. The population of M. capitata in Kāneʻohe Bay has high levels of genetic diversity, mixing, and dispersal, suggesting a capacity for acclimatization and adaptation to novel environments [ 15 ]. The specific mechanisms underlying this reproductive resilience, and the associated physiological tradeoffs, have yet to be fully defined [ 9 ]. In this study, we investigated the effects of experimental temperature-induced bleaching in M. capitata , focusing on the impact of bleaching on gametogenesis. Over a 10 month period, we monitored key physiological indicators of health, including mass spectrometry-based proteomics, to uncover the main molecular pathways underlying the phenotypic response to and recovery from temperature-induced bleaching. Proteins are essential functional molecules of cells that drive physiological and phenotypic changes in response to environmental drivers and are relevant for identifying physiological responses to changing environmental conditions. Our results uncover the diverse processes impacted by bleaching and the physiological tradeoffs of bleaching resilience. RESULTS Histology: Gamete development Corals that were experimentally bleached in September (T 1 ) 2017 had significantly smaller oocyte Feret diameter (linear mixed-effects p-value < 0.05) and a higher frequency of smaller oocytes than control coral (Fig. 1 A). At T 5 , there was no difference in the distribution of oocyte stages (Fig. 1 B) or spermatocyte stages (Fig. 1 C) between previously bleached and control corals (p > 0.05). Chlorophyll, symbionts, and lipids The amount of chlorophyll a and c 2 in coral tissue significantly differed by bleaching status and time (p < 0.05; Supplemental Table 1; Supplemental Fig. 1). Chlorophyll content (both a and c 2) generally decreased in bleached corals between T 1 and T 2 (64% and 47% reduction, respectively), followed by an increase across both bleached and control corals by T 3 (Fig. 2 B; Supplemental Fig. 1). Chlorophyll content was very similar between bleached and control corals by T 4 for chl a and T 5 for chl c 2. Symbiont densities also varied significantly by time and by treatment (Fig. 2 A, Supplemental Table 1). Symbiont counts decreased at T 2 for bleached corals. By T 4 , bleached and control symbiont counts were about equal and remained similar for the duration of the experiment. Generally, corals maintained the same genus of symbiont throughout the time series (Supplemental Fig. 2). Seven genets were dominated by Durusdinium at all time points measured; three genets had Cladocopium as the dominant symbiont for all time points. Two genets went from being Cladocopium- to Durusdinium- dominated between T 1 and T 2 , regardless of bleaching treatment. One genet changed from Cladocopium- dominance at T 1 and T 2 to Durusdinium- dominance at T 6 , regardless of bleaching status (Supplemental Fig. 2). Lipid biomass was significantly impacted by time (Fig. 2 C, Supplemental Table 1). Lipid biomass decreased dramatically in experimentally bleached coral between T 1 and T 2 but attained the same levels as paired controls by T 4 and surpassed lipid biomass in control corals at T 5 and T 6 . Lipid biomass decreased in control corals around the time of spawning, between T 5 and T 6 . Proteomics We identified 4346 proteins in the M. capitata adult proteome across all time points (Supplemental Table 2). In the results that follow, findings from the hierarchical clustering (Supplemental Fig. 3) and the PLS-DA (Fig. 3 ) are presented together. Hierarchical clustering results are referred to by cluster ID (e.g., NB17 = cluster #17 from non-bleached controls); PLS-DA protein trends are referred to by the figure panel. Supplemental Tables 2 and 3 contain details on which specific proteins were important for each analysis. Progression through gametogenesis of non-bleached control colonies Each time point in this dataset represents the proteomic profile underlying an important step in the gametogenic process. These processes are summarized in Table 1 for the control cohort. In M. capitata in Kāneʻohe Bay, the new gametogenic cycle begins between July and October [ 12 ] (Fig. 2 D), which is represented by T 1 and T 2 (September and October) in this study. PLS-DA trends B and C include proteins that increase and decrease, respectively, across early gametogenesis. This time period is marked by increasing abundance of proteins involved in lipid, protein, and carbohydrate metabolism. Many of these general metabolic themes continue through December (T 3 ), which is still within early gametogenesis for M. capitata [ 12 ]. Table 1 Summary of proteomic changes in control corals throughout gametogenesis. All processes and GO terms are elevated at the indicated time point relative to other time points. These results are summaries of the hierarchical clustering and the PLS-DA, summarized in Supplemental Fig. 3, Supplemental Tables 2 and 3, and Fig. 3 . Time Point Enriched GO Terms Important Processes Represented in Proteome T 1 and T 2 “structural constituent of cytoskeleton”; “protein phosphorylation” Lipid oxidation and metabolism; lipid binding and transport; protein turnover; immune response; carbohydrate metabolism T 3 “single-stranded RNA binding”; “semaphorin receptor binding” Citric acid cycle; protein degradation; amino acid and protein synthesis T 4 “oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen”; “cell adhesion” Extracellular matrix remodeling; skeletal mineralization; pentose phosphate pathway; microtubule organizing; transcriptional activation; steroid synthesis; protein degradation; lipid metabolism and transport; mucus T 5 “ubiquitin protein ligase activity”; “membrane protein complex”; “dynein complex” Skeletogenesis; cytoskeleton T 6 “microtubule-based movement”; “protein kinase C-activating G protein-coupled receptor signaling pathway” Cytoskeletal structure and organization; sex hormone metabolism; apoptosis; lipid metabolism In the spring, M. capitata undergo a rapid increase in oocyte size, concomitant with an increase in solar radiation (Fig. 2 )[ 12 ]. March (T 4 ) also marks a distinctive proteome change in control corals (Fig. 3 D and E). Changes in protein abundance span a diverse range of processes including extracellular matrix remodeling, transcriptional activation, and steroid synthesis. Spawning in M. capitata occurs in June and July and T 5 and T 6 capture time periods just days before the first and second spawning dates. Similar to T 4 , T 5 represents another inflection point in protein abundance, representing large changes in proteomic profile from T 4 to T 5 and from T 5 to T 6 (Fig. 3 D & E). Bleaching response and gametogenesis in experimentally bleached colonies Bleached proteomes had strikingly different temporal trends than those seen in non-bleached controls. Trends of proteomic divergence continued from bleaching through spawning with some results summarized in Table 2 . In the hierarchical clusters at the September (T 1 ) time point, there is limited overlap in the proteins that peak and are suppressed at later time points between bleached and control corals (Supplemental Fig. 3). October (T 2 ) represents a large proteomic shift in bleached corals since it is the time point that immediately followed thermal bleaching (Fig. 3 G-J). There is a dramatic change in all of the PLS-DA trends in bleached corals between T 2 and T 3 . From the hierarchical clustering, almost twice as many proteins in the bleached colonies increased in abundance at T 2 (n = 248) compared to the control colonies (n = 130), suggesting that T 2 is a critical time of recovery, supported by proteome remodeling in response to thermal stress (Supplemental Fig. 3). Elevated proteins at T 2 include some that may be biomarkers of catabolism of stored lipids since the corals remained in tanks with filtered seawater and had limited access to food via heterotrophy. Table 2 Summary of proteomic changes in bleached corals throughout bleaching recovery and gametogenesis. All processes and GO terms are elevated at the indicated time point relative to other time points. These results are summaries of the hierarchical clustering and the PLS-DA, summarized in Supplemental Fig. 3, Supplemental Tables 2 and 3, and Fig. 3 . Time Point Enriched GO Terms Important Processes Represented in Proteome T 1 “extracellular region” Retinol dehydrogenase; blood coagulation; immune response T 2 “membrane”; “signal transduction”; “methionine biosynthetic process”; “glucoronosyltransferase activity”; “regulation of hydrolase activity”; “response to oxidative stress”; “cellular oxidant detoxification” Lipid catabolism; cellular stress T 3 ATP production; extracellular matrix remodeling; lipid uptake; peptide and protein breakdown T 4 “histone binding” Mucus; lipid uptake; gametogenesis T 5 “carbohydrate derivative transport”; “RNA phosphodiester bond hydroylsis endonucleolytic” Ceramide cycle T 6 Lipid metabolism; cellular stress response; energy transfer to tissues Unlike their paired controls, the bleached corals do not have a dominant proteome signal in March (T 4 ) in the PLS-DA (Fig. 3 ), the time of rapid egg size increase (Fig. 2 D). However, T 4 represents an attainment of proteomic stasis that is maintained for many proteins through spawning (T 5 and T 6 ). In July (T 6 ), of the 177 proteins with elevated abundance in previously bleached colonies (Supplemental Fig. 3), only sixteen were also elevated in the controls. DISCUSSION The full impact of thermal bleaching on population and ecosystem scales is in part determined by the reproductive success of the corals that survive but have still experienced sub-lethal impacts of the stress. Thermal bleaching responses can be variable even within a single species from the same reef [ 16 ], suggesting that individual variability in responses can have reef-wide consequences. We followed a cohort of M. capitata through 10 months of recovery after a simulated thermal bleaching event and found that the corals surviving bleaching regain lipid reserves within two months post-bleaching and reacquire symbionts within six months, before spawning occurs. However, differences in oocyte size and protein biomarkers of certain metabolic pathways suggest that even 10 months after thermal bleaching stress, coral still express a phenotype at least partially dictated by prior thermal stress. Although natural bleaching susceptibility in M. capitata can be explained by differences in their algal symbionts [ 10 ], symbiont type was not strongly associated with the ability of coral colonies to recover from bleaching in this experiment. The focus on the impacts of bleaching on the essential process of gametogenesis in this study provides a novel perspective on thermal bleaching recovery that has not been directly addressed in previous studies on long-term impacts of bleaching using molecular markers (e.g., [ 17 ]). Reproductive success is a complicated mosaic that is determined by parent physiology and environmental conditions and culminates in larval survival and settlement; this study provides a detailed depiction of the underpinnings of reproductive success after thermal bleaching. Immediately following thermal bleaching in September, M. capitata colonies lost their symbionts and much of their lipid reserves (Fig. 2 A & 2 C). It is well established that thermal stress can disrupt the symbiotic relationship between the coral and its dinoflagellate symbionts (Symbiodiniceae) (e.g., [ 18 ]) and once the symbionts are expelled and/or digested, the host loses an important metabolic resource. Symbiont genus did not impact bleaching recovery in this study, as has been observed in natural, field-based bleaching studies (i.e. [ 10 ]). We hypothesize that symbiont genus had no impact on bleaching recovery because we intentionally induced complete bleaching in corals hosting varying symbiont community types to generate our bleached cohort, but additional work is needed to distinguish the relationship between thermal resistance and recovery dynamics. There are proteomic signals detected in this study of symbiont rejection and reacquisition in the corals that underwent experimental bleaching. The GS/GOGAT (glutamine synthase/glutamine oxoglutarate aminotransferase) system is the dominant means of N acquisition in a healthy holobiont [ 19 ]; this system breaks down when the symbiotic relationship is disrupted and is in part replaced by host urease expression to acquire N [ 20 ], [ 16 ]. In October (T 2 ), there was a decrease in abundance of coral glutamine synthetase (part of the GS/GOGAT system) and an increase in urease enzymes relative to control corals, suggesting a shift in host N acquisition reflecting the loss of photosynthate (Fig. 4 , Supp. Figure 4 ) [ 16 ]. Coral N acquisition via symbiont pathways is still suppressed in December (T 3 ), reinforcing that symbionts are not yet providing all the resources the host needs (Fig. 4 , Supp. Figure 4 ). Isocitrate dehydrogenase is another enzyme associated with GS-GOGAT and may be a biomarker of symbiotic dysfunction; some homologs of this enzyme are elevated in bleached compared to control colonies in December (T 3 ) and March (T 4 ) (Supp. Figure 4 ). Thus, during this critical period of early gametogenesis, the previously bleached corals are relying predominantly on heterotrophic feeding to rebuild lipid reserves. Symbiont reacquisition was almost complete by March (T 4 ) in previously bleached coral and abundances are about equal with their non-bleached paired controls. In the proteome, there was an almost eight-fold decrease in Rab11a abundance in March (Fig. 4 ), a protein that is suppressed during healthy symbiosis [ 21 ]. Healthy, photosynthesizing symbionts would provide the coral host metabolic by-products, such as simple carbohydrates, which the host then metabolizes [ 22 ]. Two metabolic enzymes (pyranose oxidase and succinate-semialdehyde dehydrogenase), which may be involved in processing of symbiont-derived resources, decrease post-bleaching and rebound through symbiont reacquisition, perhaps signaling a reestablishment of symbiont byproduct-derived carbohydrate metabolism (Supp. Figure 4 ). During physiological stress, organisms may reallocate energetic resources to respond to the stress while maintaining essential physiological functions. In corals, skeletogenesis decreases immediately following bleaching stress or reduction in photosynthate (e.g., [ 7 ]). In the M. capitata proteome of experimentally bleached corals, we see a suppression in the abundance of all isoforms of carbonic anhydrase, a skeletogenesis protein, suggesting a decrease in skeletal growth relative to non-bleached controls (Fig. 4 , Supp. Figure 4 ), which has been previously observed [ 5 ]. This trend in carbonic anhydrase abundance is recapitulated in the M. capitata transcriptome post-thermal stress [ 23 ]. Another aspect of the coral response to bleaching is an increase in reactive oxygen species (ROS) and a concomitant increase in ROS scavengers and repair proteins to mitigate and respond to cellular damage [ 23 ], [ 24 ]. As in [ 24 ], homologs of glutathione-S-transferase and caspase were elevated in bleached corals compared to non-bleached controls in October, suggestive of a ROS response (Fig. 4 , Supp. Figure 4 ). ROS response proteins elevated in October (T 2 ) also include peroxidasin (a known inhibitor of ROS), as well as proteins that are likely responding to the consequences of cellular damage: protein disulfide isomerase A6 (inhibits aggregation of misfolded proteins) and leukocyte elastase inhibitor (protects cells from proteases released into the cytoplasm during stress). These proteins all potentially represent a cellular effort to repair and mitigate damage to DNA, proteins, and other cellular structures in the aftermath of thermal stress. The timing of symbiont loss, which corresponds to when most natural bleaching events occur, overlaps with the onset of gametogenesis in M. capitata [ 12 ]. Gametogenesis is an energy intensive process, and the combined effect of lipid store catabolism with the loss of symbiont resources could conceivably deplete the energy store necessary to form viable gametes. October (T 2 ) also represents the start of a months-long recovery period for bleached corals. During these months post-bleaching, we show evidence that bleached coral survive bleaching by first catabolizing lipid reserves (Figs. 2 C, 4 , Supp. Figure 4 ) and then switching to heterotrophic feeding (Fig. 4 , Supp. Figure 4 ) to rapidly restore lipid levels (Fig. 2 C) while reacquiring symbionts (Fig. 2 A) and acquire enough autotrophically derived C before spawning (Fig. 2 A, 4 , Supp. Figure 4 ) to successfully undergo gametogenesis, with potential tradeoffs in egg size (Fig. 1 A). Lipid and proteomic data in the present study suggest that bleached corals began to catabolize their lipid reserves post-bleaching to make up for the loss of metabolic products from their symbionts. The rapid mobilization of stored lipids to compensate for reduced photosynthate after bleaching has been well documented in M. capitata [ 25 ], [ 17 ], [ 9 ] and other coral species [ 18 ], [ 22 ]. Many protein biomarkers detected at this time point also confirm a probable up-regulation of lipid catabolism immediately post-bleaching (Fig. 4 and Supplemental Fig. 4). Lipids are essential macromolecules in the development of gametes in marine organisms (e.g., [ 26 ]) and catabolism of lipids for other physiological purposes may have lasting impacts on the gametogenic process. The experimentally bleached corals depleted lipid reserves through the first two months post-bleaching (Fig. 2 C), with proteomics revealing some of the molecular mechanisms of lipid catabolism during this time. Phospholipase B is one of the proteins that follows a trend of elevated abundance in bleached coral compared to non-bleached controls in October (T 2 ) [ 24 ] (Fig. 4 ; Supplemental Fig. 4). Even if symbionts remain in the bleached corals, thermally stressed symbionts may provide fewer lipids to their host [ 24 ]. Therefore, the lipid metabolism enzymes that are elevated at this time point are either acting on 1) the few lipids provided by the remaining symbionts, 2) heterotrophically-derived lipids, or 3) storage lipids from the coral tissue. M. capitata use stored lipid reserves when photosynthate and heterotrophy are unavailable [ 25 ], while as much as 70% of M. capitata lipids can be heterotrophically derived immediately post-bleaching [ 17 ]. In October, the corals had not yet been outplanted to racks on the reef and therefore had access to only limited suspended plankton in tanks for heterotrophy. Given the low number of symbionts and rapid decrease in lipid reserves, the corals likely derived energy from their stored lipids at T 2 . Lipids are the main energy source that corals allocate to their gametes [ 27 ] and the depletion of these stores in early gametogenesis impacts gamete production. In December, bleached corals had not yet reacquired all their symbionts, yet lipid content was almost on par with their non-bleached control counterparts. This rapid reacquisition of lipids has been previously observed in M. capitata and may be in part attributable to more efficient resource conservation due to a lower respiration rate than other coral species [ 25 ], as well as heterotrophic feeding. In December, many lipid hydrolysis enzymes, potentially linked to food acquisition and lipid digestion, were at relatively high abundance in bleached coral compared to non-bleached controls (Fig. 4 , Supp. Figure 4 ). Some proteins involved in peptide and protein breakdown were also elevated, and two proteins involved in ATP synthesis increased in abundance from October (T 2 ) through March (T 4 , Fig. 4 , Supp. Figure 4 ). Combining all the molecular evidence, it suggests that M. capitata engages in external food acquisition and breakdown, as well as cellular energy production despite low levels of symbionts. M. capitata its known for its trophic plasticity and can acquire the food necessary to fulfill its daily metabolic needs with heterotrophy alone [ 25 ]. This flexibility in the nutrient acquisition is not present across all coral species [ 25 ] and may be part of the phenotype of increased bleaching resilience observed in M. capitata [ 25 ]. Between October (T 2 ) and December (T 3 ), bleached M. capitata are likely relying on heterotrophy almost exclusively to rebuild lipid reserves, which could provide energy to support spawning and/or survival during a future bleaching event. March (T 4 ) represents a peak in lipid biomass for these corals since lipids are allocated to oocytes, and adult tissue lipid levels decline post-spawning [ 28 ], [ 29 ]. This period of lipid accumulation leading up to allocation to oocytes is essential for embryo and larval development and survival in their planktonic phase [ 30 ]. Some lipid metabolism enzymes were relatively lower in bleached corals in March (e.g., lipase, phospholipase D3, saposin domain-containing protein), while others were relatively elevated (e.g., phospholipase A2 and B, prosaposin). Two hypotheses for these trends could be that 1) the bleached coral have \"overcompensated\" for the depletion of lipid reserves during bleaching and have upregulated their lipid metabolism and storage pathways via increased heterotrophy and/or the return of symbiosis coinciding with increased light and temperature during spring months. Alternatively, 2) bleached coral may be dedicating relatively less lipid to their oocytes and thus maintaining a higher reserve in their somatic tissue than controls. The elevated level of lipids in adult coral is likely due to M. capitata 's ability to store heterotrophically derived carbon, as suggested by [ 31 ], and as evidenced by the trends seen in early lipid reacquisition in this study. The June (T 5 ) and July (T 6 ) time points represent the time periods just before spawning events and are marked in control coral by a decrease in lipid biomass (Fig. 2 C) and another inflection point in trends of protein abundance in the PLS-DA trends (Fig. 3 D & 3 E). The dip in lipid biomass is not observed in bleached corals (Fig. 2 C); higher lipid levels may help these corals survive future bleaching events [ 18 ]. The dip in lipid levels of adult tissues is a consistent trend across broadcast-spawning corals that marks the allocation of lipids to oocytes [ 7 ], [ 27 ]. It has been hypothesized that the drop in tissue lipid content with spawning is directly correlated with energy allocation to gametes [ 7 ]. Even though bleached M. capitata prioritize energy transfer to oocytes [ 9 ], the lack of early access to autotrophic C may have resulted in less energy allocation to gametes in bleached corals, compared to controls. In the proteome, homologs of vitellogenin, a major egg yolk protein, are all at lower abundance in previously bleached coral compared to non-bleached controls in June, suggesting less lipid allocation to oocytes (Supp. Figure 4 ). Repeated bleaching events can lead to increased mortality in some species, perhaps because energetic reserves used to survive prior stressful events are depleted [ 32 ]. M. capitata in the dynamic Kāneʻohe Bay may have evolved physiological mechanisms to survive repeated environmental stresses (e.g. [ 33 ]). The tradeoff, however, could have implications for larval viability if there are less maternal lipids to sustain the non-feeding stages of development and to maintain positive buoyancy [ 29 ]. While the dominant protein abundance trends in bleached corals were in response to bleaching, a significant proteomic shift occurred in non-bleached controls during March (T 4 ), coinciding with the period of most rapid oocyte size increase and onset of spermatogenesis (Fig. 3 D, Fig. 5 E & 5 F). A wide range of functions are represented by the proteins of control corals that peak in abundance in March. The proteins encompass functions in DNA and cellular replication, protein turnover, and signaling via neurons and neuropeptides (Fig. 4 , Supp. Figure 4 ). Signals of extracellular matrix reorganization, a basal process essential to gametogenesis [ 34 ], were detected in the control corals: two proteins that negatively regulate or degrade the ECM were at decreased abundance in March relative to previous time points (Fig. 4 , Supp. Figure 4 ). These proteins represent the complex physiological processes necessary to support gametogenesis. As in other animals, sex hormones regulate reproductive maturation in corals (e.g., [ 35 ]) and we identified four proteins with likely sex hormone regulation functions that are impacted by thermal bleaching. The hierarchical clusters that peak in March (Supp. Figure 3 ) include 1) steroid 17-alpha-hydroxylase (also known as CYP17A1), a protein important in regulation of sex hormones during gametogenesis [ 36 ] (Fig. 4 , Supp. Figure 4 ). CYP17A1 abundance is not impacted by bleaching recovery at the time points investigated. 2) Steroidogenic acute regulatory protein (StAR) abundance is depressed in previously bleached corals at T 2 and T 3 and then increases to levels higher than those in controls at T 5 . StAR is an essential early step in steroid synthesis and has been identified in other corals genomes [ 35 ] (Supp. Figure 4 ). 3) Protein flightless-1 and 4) 17B hydroxysteroid dehydrogenase are both at relatively low abundance in bleached corals at T 4 . The former may regulate transcription downstream of estrogen and androgen receptors, and the latter is known to regulate estrogen and androgen levels in mammals and has known estrogen activity in corals [ 37 ] (Supp. Figure 4 ). In the weeks leading up to spawning, sex hormones increase in M. capitata [ 23 ] and the proteins regulating these hormones detected in this study give further insight into patterns of sex hormone production during gametogenesis. In June (T 5 ), oocyte and spermatocyte stages were similar between bleached and control coral, but oocyte Feret diameter was significantly smaller in previously bleached corals (Fig. 1 ). Oocyte diameter reduction is a common effect of bleaching across coral species [ 38 ]. Even though bleached coral had attained the same lipid levels as controls by December, these lipid stores are likely derived mostly from heterotrophically derived carbon since symbiont levels were still low. M. capitata colonies do not allocate heterotrophically derived carbon to oocytes, only autotrophic carbon [ 29 ], [ 9 ]. Previously bleached corals likely had less autotrophically derived C for their oocytes than controls. M. capitata and other corals provision eggs with lipids, proteins, symbionts, and photoprotectant molecules [ 29 ], [ 30 ]; adult bleaching can reduce the amounts of these molecules allocated to oocytes [ 28 ], which may be reflected in the smaller size of bleached corals’ oocytes in this study. Bleaching impacts on coral gametogenesis vary both across species and populations within a species. If a coral population is highly tolerant to bleaching stress, they may still be able to execute full gametogenesis as seen in this study and others (e.g. [ 38 ]). Bleaching tolerance may be species-specific or genetically determined but may also be a result of acclimatization over multiple bleaching events (e.g., [ 39 ]). The severity of the bleaching event and/or a history of thermal bleaching exposure can dictate whether and to what degree a coral can achieve gametogenesis [ 38 ]. M. capitata in Kāneʻohe Bay experienced thermal stress and may have bleached in 2014 and 2015 (two and three years before this study, respectively; [ 8 ]) and seem to have evolved resilience to disturbances [ 33 ]. Their combined genetic and ecological histories may give them the physiological capacity to accomplish gametogenesis despite bleaching, while other species or populations may fail. Some of the functional protein biomarkers identified here may help identify essential traits and mechanisms of thermal bleaching-resilient phenotypes. M. capitata demonstrated a potential for resilience to thermal bleaching stress as well as an ability to rapidly recover symbionts and lipid levels post-bleaching. However, the bleached colonies did not have access to symbiont-provided autotrophic carbon during the critical months of early gametogenesis, which likely resulted in a time lag in upregulating some of the physiological processes necessary for gametogenesis (Fig. 4 ). One result of this delay in resource access was significantly smaller oocytes in bleached corals. Corals in Kāneʻohe Bay have experienced increasingly frequent thermal bleaching events in recent years, the effects of which may be seen in smaller oocytes across the population when compared with oocyte sizes from previous years (e.g., [ 40 ], [ 14 ]). Additionally, previous work has suggested that the impacts of bleaching can be long-lasting and more detrimental for coral sperm than for eggs [ 41 ]. A longer-term study that follows these corals across years and multiple bleaching events would clarify whether these strategies facilitate faster recovery from bleaching events. Additionally, it is important to better understand if there are any short- or long-term population-level consequences in allocating less of the adult lipid biomass to gametes in terms of the number of viable embryos and successfully settled juveniles. Since both adult coral survival and juvenile settlement success are integral to continued reef survival, it is not enough that we understand if corals survive thermal bleaching; we also must understand the cost of that survival. METHODS Coral Selection and Bleaching Details for coral collection and historical bleaching regime can be found in [ 11 ]. Seventy-four M. capitata coral (approximately 30 cm in diameter) were collected from patch reefs located in Kāneʻohe Bay, Oʻahu, Hawaiʻi around the Hawaiʻi Institute of Marine Biology (HIMB, 21.428°N, 157.792°W) in August 2017. Each coral was further divided in half so that genetically identical halves (i.e. ramets of a genet) were replicated in the control and treatment conditions (Fig. 5 ). Experimental corals were acclimated in flow-through outdoor tanks (three tanks per treatment group) for 7–10 days. In September (T 1 ), the period when bleaching events typically occur in Hawaiʻi [ 8 ], corals in the bleaching treatment were exposed to warmer water temperatures (30°C) to simulate these conditions (Supplemental Fig. 5). Experimental corals were then placed on racks off HIMB after the sampling time point on October 1, 2017 (T 2 ) to monitor their recovery and physiological response to the in situ environment through July (T 6 ) (Supplemental Fig. 6). Mortality and bleaching assessments were conducted weekly using the Coral Watch Coral Health Chart [ 42 ]. The time points when corals were sampled are: 1) at the end of August (August 30, 2017) after corals acclimated in the tanks but before they were bleached (T 1 ); 2) in October, 24 hours after bleached corals were returned to ambient temperature (T 2 ; October 1, 2017); 3) in December (T 3 ; December 20, 2017); 4) in March, during the period of the rapid increase in egg size (T 4 ; March 29, 2018) [ 12 ]; 5) in June, at the beginning of the spawning season, when symbionts are vertically transferred to gamete bundles (T 5 ; June 8, 2018) [ 29 ]; 6) and in July, later in the spawning season (T 6 ; July 9, 2018). Additional details are provided in Supplemental Methods. Histology Histology slides were made from serial sections of M. capitata tissue samples collected in June 2018 (T 5 ). Slides were stained with hematoxylin and eosin before observation under a compound microscope, photographed using a camera attachment (model DS-Fi3, Nikon Instruments Inc.), and analyzed with NIS-Elements imaging software. Oocyte measurements were made in Image-J software version 1.52 using Feret’s Statistical Diameter to estimate size. Only oocytes with visible nuclei were measured to ensure standardization of measurements along the widest axis of the oocyte. Developmental stage of gametes was assessed using morphological guidelines and Feret diameter size ranges [ 12 ]. Although the response variable, Feret diameter, was not normally distributed (Fig. 2 A), the sample size was sufficiently large (n = 858) to fulfill assumptions of the Central Limit Theorem that the sampling distribution is approximately normally distributed. Means, standard deviations, and ranges of oocyte Feret diameter were calculated for both control and experimentally bleached coral. Feret differences and oocyte and sperm stages between bleached and control corals were evaluated following [ 43 ]. Additional details are provided in Supplemental Methods. Chlorophyll, symbiont counts, symbiont clades, and lipids Chlorophyll a (Chl a ) and dinoflagellate symbiont (Symbiodiniceae) counts were analyzed as reported by [ 11 ]. Briefly, Chl a was extracted on ground whole coral (tissue and skeleton) samples using 100% acetone [ 44 ], determining absorbance at 630, 663, and 750 nm, and standardized to total dry tissue weight. For symbiont counts, dinoflagellate symbionts were separated from ground whole coral samples; isolated pellets were resuspended in filtered seawater with 1% formalin and 2–3 drops of Lugol’s iodine. Three subsamples were counted using a hemocytometer, and the mean count was reported. Relative symbiont abundances (genera Cladocopium and Durusdinium ) were measured with qPCR for tissue samples taken in September, October, and July (T 1 , T 2 , and T 6 DNA was extracted from tissue using a CTAB-chloroform protocol ( dx.doi.org/10.17504/protocols.io.dyq7vv )). Proportions of Cladocopium and Durusdinium symbiont cells in each sample were quantified with qPCR using actin assays [ 45 ] on an Agilent AriaMX system with two technical replicates run for 40 cycles. Additional details are provided in Supplemental Methods. Lipids were extracted from ground whole coral samples with a 2:1 chloroform: methanol solution, 0.88% KCl and 100% chloroform washes [ 46 ]. Extracted lipid samples were dried to a constant weight under grade 5.0 N 2 gas and standardized to total dry tissue weight. Linear mixed effects models (lmer in the lme4 package [ 47 ]) were applied to determine significant differences in chlorophyll a and c2 concentration, symbiont counts, and total lipids with bleaching status and time as fixed effects and coral genet as a random effect. Significant effects (p < 0.05) were determined in lmerTest [ 48 ]. There was not enough data for the symbiont genera to fit a generalized linear model, but qualitative results are described. Proteomics Protein digestions were conducted with 100 µg of protein per coral sample as described in [ 11 ]. M. capitata samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC‒MS/MS) on a Q‒Exactive‒HF (Thermo) in Data Dependent Acquisition (DDA) mode. From each mass spectrometry experiment, M. capitata peptides were identified and proteins were inferred using a proteome derived from the M. capitata genome (NCBI Bioproject Accession no. PRJNA509219) [ 49 ]. The M. capitata proteome was concatenated with a predicted proteome for Symbiodiniaceae genus Cladocopium ( http://symbs.reefgenomics.org/download/ ) and 50 common contaminants (cRAPome [ 50 ]); the final proteome database contained 99,221 sequences. The raw MS data (PRIDE accession PXD021262, reviewer username = [email protected] , password = r5h4H1vo) were searched against the protein database using Comet v 2019.01 rev.5 [ 51 ]. Concatenated target–decoy database searches were followed by PeptideProphet and ProteinProphet [ 52 ] with a probability cut-off of 0 to allow for FDR cut-off downstream in Abacus. Resulting data files across all samples were analyzed with Abacus [ 53 ] to generate consistent protein inferences across replicates and to calculate normalized spectral abundance factors (NSAF) with an FDR cut-off of 0.01 (protein probability of 0.91). Proteins were included in downstream analyses if two unique peptides were identified across all mass spectrometry experiments and if they were not flagged as outliers [ 54 ]. Proteins important to experimental bleaching response and/or to gametogenesis were identified with two main methods described below: 1) hierarchical clustering and 2) discriminant analysis by partial least squares (PLS-DA). These two methods are complementary and were used to identify which molecular processes change over time and how those changes may impact bleaching recovery and the timeline of gametogenesis. Additional proteomics details are provided in Supplemental Methods. All analyses described above were accomplished in R [ 55 ]. All data files and R code used for analyses are available on Dryad: https://datadryad.org/stash/share/TH40Lri9NsCmVVGpYe92iJ2jTfVsvJT0ryORkPSba70 Declarations Author Contribution ETS performed the protein extractions, data analysis and interpretation, and was the primary author of the manuscript. ED contributed to data analysis and interpretation. TB was responsible for all field experiments and assisted with lab work. JBA assisted with field work and symbiont counts. CHB did the symbiont counts. MR contributed to data analysis. JD and CDK performed the qPCR of the symbiont genera. LJR contributed to funding acquisition, study design, field experiments, and performed the lipid analyses. BLN contributed to the study design, protein extractions, and performed the mass spectrometry analysis. JLPG conceived of the experimental time-series study design and led the field work and project management. All co-authors contributed to writing of the manuscript. Acknowledgement This research was supported by the National Science Foundation’s Division of Integrative Organismal Systems, Integrative Ecological Physiology Program (NSF IOS-IEP) 1655682 to JP-G and BN, NSF IOS-IEP 1655888 to LJR and NSF CAREER (BIO-OCE, 2044840) and the Sloan Foundation Fellowship to JP-G, and start-up funding from the University of Southern California to CDK. Additional support came from the University of Washington's Proteomics Resource (UWPR95794). We thank Dr. Ruth Gates and the Gates Coral Laboratory for our sponsorship at the Hawai’i Institute of Marine Biology. We thank G. Kreitman, M. Jaffe, S. Frangos, J. Davidson, and E. Lenz, for field and laboratory support. We would also like to acknowledge the essential work done by the Genome Sciences IT group to support all our work. ETS would like to thank IJE and EGE for continuing inspiration. Data Availability The raw proteomics mass spectrometry files are available on PRIDE under accession PXD021262, reviewer username = [email protected] , password = r5h4H1voAll R code and files necessary for the statistical analyses and plots presented in this manuscript are available on Dryad: https://datadryad.org/stash/share/TH40Lri9NsCmVVGpYe92iJ2jTfVsvJT0ryORkPSba70 COMPETING INTERESTS The authors have no competing interests to declare. References Hughes, T.P., Baird, A.H., Bellwood, D.R., Card, M., Connolly, S.R., Folke, C., Grosberg, R., Hoegh-Guldberg, O., Jackson, J.B.C., Kleypas, J., et al. Climate change, human impacts, and the resilience of coral reefs. Science 30, 929–933 (2003). Donelson, J.M., Wong, M. Booth, D.J., & Munday, P.L. Transgenerational plasticity of reproduction depends on rate of warming across generations. Evolutionary Applications 9, 1072–1081 (2016). Wernberg, T., Coleman, M.A., Bennett, S., Thomsen, M.S., Tuya, F., Kelaher, B.P. 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Supplementary Files SuppFig1Mcapchlc2Jan2023.png SuppFig2symbiontcladesRandSv2.png SuppFig3hierarchicalclusters.pdf Suppfig4proteinplots.pdf SuppFig5tankstemplight.png SuppFig6rackstemp.png SuppFig7partialmortality.pdf SuppTable1lmresultscoral.xlsx SuppTable2proteinannotationsandhierarchicalclusterassignments.txt SuppTable3PLSDAproteinannotations.txt SupplementalMethods.docx Cite Share Download PDF Status: Published Journal Publication published 13 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Aug, 2024 Reviews received at journal 22 Jul, 2024 Reviews received at journal 21 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviewers invited by journal 04 Jul, 2024 Editor assigned by journal 01 Jul, 2024 Editor invited by journal 26 Jun, 2024 Submission checks completed at journal 22 Jun, 2024 First submitted to journal 10 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Rodrigues\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Villanova University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"LF\",\"middleName\":\"\",\"lastName\":\"Rodrigues\",\"suffix\":\"\"},{\"id\":323645613,\"identity\":\"ccb6b418-d61a-4b05-b01b-24a7862dfabf\",\"order_by\":9,\"name\":\"BL Nunn\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Washington\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"BL\",\"middleName\":\"\",\"lastName\":\"Nunn\",\"suffix\":\"\"},{\"id\":323645617,\"identity\":\"c57b6223-4804-4f7c-86b5-2725049759db\",\"order_by\":10,\"name\":\"JL Padilla-Gamiño\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Washington\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"JL\",\"middleName\":\"\",\"lastName\":\"Padilla-Gamiño\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-06-10 20:08:18\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4559943/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4559943/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-024-78768-7\",\"type\":\"published\",\"date\":\"2024-11-13T15:58:01+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":60124956,\"identity\":\"8a17b92f-7603-412c-aecf-a872693f1cce\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:22\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":62972,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe distribution of coral oocyte Feret diameters in June 2018 (T\\u003csub\\u003e5\\u003c/sub\\u003e) for previously bleached (blue) and non-bleached control (brown) coral colonies (A). Vertical lines represent the mean Feret diameter for each group, which were significantly different from each other. Oocyte stage distributions for bleached and control coral from June 2018 (T\\u003csub\\u003e5\\u003c/sub\\u003e) demonstrated no significant differences (B). Spermatocyte stage distributions for previously bleached and control coral were also not significantly different (C). The coral genets for the histology analyses are not the same as the ones used for proteomics.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1SciReports.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/68825943d79598c6d504f471.png\"},{\"id\":60124954,\"identity\":\"fa9a1885-850d-4a9d-8ede-bad860b26c32\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:22\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":281047,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSymbiont counts (A), chlorophyll \\u003cem\\u003ea\\u003c/em\\u003e (B), lipid biomass (C), and average egg diameter of \\u003cem\\u003eM. capitata \\u003c/em\\u003efrom Kāneʻohe Bay in 2009 adapted from [12] (D) across time. For symbiont counts, chlorophyll \\u003cem\\u003ea\\u003c/em\\u003e, and lipids, all measured in the current 2017/2018 study, values from bleached corals are in blue and non-bleached controls are in brown. Points represent average values with standard error bars. The gray point represents the value for T\\u003csub\\u003e1\\u003c/sub\\u003e (before thermal stress) with the bleaching event marked with an orange dotted line. The 2009 egg diameter data (with standard deviation represented as gray vertical lines) serves to situate the progression of thermal bleaching recovery within the context of the gametogenic cycle.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2SciReports.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/111b5ba91ba406cfb4553283.png\"},{\"id\":60125493,\"identity\":\"c1645c89-a2ac-420a-a19a-de70726ddb86\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:48:22\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":896584,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePlots of the proteins identified with discriminant analysis of partial least squares (PLS-DA), including NMDS of the top 100 proteins that contribute to PLS-DA variance for non-bleached controls (A) and the top 100 proteins for bleached corals (F) across all time points; and line plots of the abundance trends over time for each cluster for control (B-E) and bleached corals (G-J). Details on these proteins can be found in Supplemental Table 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3SciReports.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/2a14e56763444bcf6a2b093f.png\"},{\"id\":60124957,\"identity\":\"4b464efc-c4b8-4c44-8fcb-6fc90b970541\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:22\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1163950,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA conceptual timeline of protein abundance changes in bleached (bottom timeline) and non-bleached control (upper timeline) corals. Physiological processes that are elevated for bleached or control corals are listed for each time point along the timeline (blue and brown lines, respectively). For some of the processes, representative plots of protein abundance are indicated below. Additional proteins are shown in similar plots in Supplemental Figure 4.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4SciReports.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/31e3a2bc6aa512027718c076.png\"},{\"id\":60125495,\"identity\":\"9b5e5b41-594a-4d44-a2a6-e3b14f1b8c27\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:48:22\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":556972,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExperimental design and bleaching status of \\u003cem\\u003eM. capitata \\u003c/em\\u003eexposed to thermal stress. September (T\\u003csub\\u003e1\\u003c/sub\\u003e; pre-bleaching) through March (T\\u003csub\\u003e4\\u003c/sub\\u003e; visual recovery from bleaching, based on Coral Watch Coral Health Chart categories). Corals were also sampled in June (T\\u003csub\\u003e5\\u003c/sub\\u003e) and July (T\\u003csub\\u003e6\\u003c/sub\\u003e) during the spawning season and their colors remained unchanged during this time period. The period of gametogenesis begins in October (T\\u003csub\\u003e2\\u003c/sub\\u003e) and progresses through a period of rapid increase in oocyte size (March, T\\u003csub\\u003e4\\u003c/sub\\u003e) until spawning in June or July (T\\u003csub\\u003e5\\u003c/sub\\u003e and T\\u003csub\\u003e6\\u003c/sub\\u003e).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5SciReports.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/3af3619e98ecb62b0bf1179a.png\"},{\"id\":69286045,\"identity\":\"f9a25bee-54bf-4730-b136-953dd4f11f55\",\"added_by\":\"auto\",\"created_at\":\"2024-11-18 19:29:13\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3975090,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/d844d1fa-dff9-4ad6-8fa1-0058515ed7e9.pdf\"},{\"id\":60125494,\"identity\":\"136c1b81-f0a2-498b-91cd-17279d636d3e\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:48:22\",\"extension\":\"png\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1733102,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppFig1Mcapchlc2Jan2023.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/982321b785a48e8834d49c12.png\"},{\"id\":60124959,\"identity\":\"e09b9745-4e9a-493f-90e1-513d4c4263b3\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 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05:48:22\",\"extension\":\"png\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2835565,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppFig6rackstemp.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/cef4d35ea7054ff9a33decef.png\"},{\"id\":60124962,\"identity\":\"4188e384-8a23-4bcd-b79e-8c128ec1bdba\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:22\",\"extension\":\"pdf\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":311935,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppFig7partialmortality.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/2511f1ca77c8e19d3cd0cef2.pdf\"},{\"id\":60124968,\"identity\":\"cb705e18-a814-4197-b944-ad0f73b5267b\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:23\",\"extension\":\"xlsx\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":10480,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppTable1lmresultscoral.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/321165b9ac4e312191e87c67.xlsx\"},{\"id\":60124964,\"identity\":\"c7467d12-3c51-4322-a3cb-b0a056b0ef38\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:22\",\"extension\":\"txt\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2194362,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppTable2proteinannotationsandhierarchicalclusterassignments.txt\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/fccb02865eb475111b78c489.txt\"},{\"id\":60124989,\"identity\":\"02149ce1-ab4e-4f42-b7a7-425fe371f7c8\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:40:24\",\"extension\":\"txt\",\"order_by\":10,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":31952,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SuppTable3PLSDAproteinannotations.txt\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/411b0f93bda9af3c04c73fd7.txt\"},{\"id\":60125496,\"identity\":\"767f057c-ceab-4276-86ea-cf5336e05356\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 05:48:22\",\"extension\":\"docx\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":17716,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalMethods.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4559943/v1/ec865c5df9a0fc8819249e0b.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Reproductive Resilience: Pathways to Gametogenic Success in Montipora capitata after Bleaching \",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eReproduction is essential for species propagation and potential evolutionary adaptation, both of which are critical for species survival amid the unprecedented challenges of a rapidly changing climate. Worldwide coral reefs are experiencing increased frequency and severity of conditions related to climate change and human activity, such as increased sea surface temperatures, ocean acidification, sedimentation, and pollution (reviewed in [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]). Continuation of successful reproduction under these conditions will likely require both extensive physiological plasticity (e.g. [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]) as well as high genetic diversity (e.g. [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]) to enable adaptation to novel environments. Despite the importance of reefs to coastal ecosystems and economies (e.g.,[\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]), the underlying physiological acclimatization needed for continued resilient and successful reproduction in corals experiencing environmental stress are not known.\\u003c/p\\u003e \\u003cp\\u003eSuccessful reproduction despite experiencing physiological stress may come at a cost to the reproducing adult. If gametogenesis is energetically prioritized in a resource-limited physiological state, such as after thermal bleaching, colony growth [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e] or even survival and susceptibility to disease [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e] may be at risk. This balance between the long-term benefits of reproduction versus the short-term tradeoffs in growth and health may also have a tipping point. In the coral \\u003cem\\u003eMontipora digitata\\u003c/em\\u003e, a reduction in photosynthesis led to an energetic shift, prioritizing of gametogenesis to a certain extent. Partially shaded colonies allocated a larger percentage of their available energy to gametogenesis and less to colony growth, whereas fully shaded colonies ceased gametogenesis completely [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. This suggests that the phenotypic plasticity has limits and beyond these limits short-term colony survival is prioritized over reproduction.\\u003c/p\\u003e \\u003cp\\u003eBleaching is a stress response associated with a variety of environmental stressors that occur when a coral colony no longer maintains its symbiotic relationship with algal endosymbionts and appears pale or white in color. In Hawaiʻi, thermal bleaching typically occurs in August or September when seawater temperature peaks [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], coinciding with the beginning of a new gametogenic cycle for \\u003cem\\u003eMontipora capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Various aspects of bleaching recovery in \\u003cem\\u003eM. capitata\\u003c/em\\u003e have been investigated (e.g., [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]), but specific mechanisms facilitating continued gametogenesis post-bleaching have not been investigated. Specifically, the question of how corals overcome the physiological tax of bleaching at essential points in the gametogenic process to realize full gametogenesis has never been addressed. Uncovering the tradeoffs necessary for gametogenesis post-bleaching could give insight into what is required for population persistence in a changing ocean.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eM. capitata\\u003c/em\\u003e is a simultaneous hermaphrodite with gametogenesis lasting the better part of a year [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Oogenesis begins in late summer/early fall and progresses through a period of rapid oocyte size increase in the spring until spawning in June or July [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Spermatogenesis is a shorter process, lasting four to five months, from April to June/July [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. M. \\u003cem\\u003ecapitata\\u003c/em\\u003e has previously shown resilience in its gametogenic process in the face of environmental stress (e.g. [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]). Its long gametogenic cycle could make \\u003cem\\u003eM. capitata\\u003c/em\\u003e more vulnerable to disruptive environmental impacts, but may also provide more time for coral recovery and spawning. The latter seems to be the case for \\u003cem\\u003eM. capitata\\u003c/em\\u003e, as the species has previously demonstrated high phenotypic canalization in gametogenesis in response to a variety of environmental drivers, such as ammonium enrichment [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e], sedimentation [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], and thermal stress [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Resilience in reproduction is essential to overall population survival in changing environments. The population of \\u003cem\\u003eM. capitata\\u003c/em\\u003e in Kāneʻohe Bay has high levels of genetic diversity, mixing, and dispersal, suggesting a capacity for acclimatization and adaptation to novel environments [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. The specific mechanisms underlying this reproductive resilience, and the associated physiological tradeoffs, have yet to be fully defined [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn this study, we investigated the effects of experimental temperature-induced bleaching in \\u003cem\\u003eM. capitata\\u003c/em\\u003e, focusing on the impact of bleaching on gametogenesis. Over a 10 month period, we monitored key physiological indicators of health, including mass spectrometry-based proteomics, to uncover the main molecular pathways underlying the phenotypic response to and recovery from temperature-induced bleaching. Proteins are essential functional molecules of cells that drive physiological and phenotypic changes in response to environmental drivers and are relevant for identifying physiological responses to changing environmental conditions. Our results uncover the diverse processes impacted by bleaching and the physiological tradeoffs of bleaching resilience.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cp\\u003eHistology: Gamete development\\u003c/p\\u003e \\u003cp\\u003eCorals that were experimentally bleached in September (T\\u003csub\\u003e1\\u003c/sub\\u003e) 2017 had significantly smaller oocyte Feret diameter (linear mixed-effects p-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and a higher frequency of smaller oocytes than control coral (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). At T\\u003csub\\u003e5\\u003c/sub\\u003e, there was no difference in the distribution of oocyte stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB) or spermatocyte stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC) between previously bleached and control corals (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05).\\u003c/p\\u003e \\u003cp\\u003eChlorophyll, symbionts, and lipids\\u003c/p\\u003e \\u003cp\\u003eThe amount of chlorophyll \\u003cem\\u003ea\\u003c/em\\u003e and \\u003cem\\u003ec\\u003c/em\\u003e2 in coral tissue significantly differed by bleaching status and time (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Supplemental Table\\u0026nbsp;1; Supplemental Fig.\\u0026nbsp;1). Chlorophyll content (both \\u003cem\\u003ea\\u003c/em\\u003e and \\u003cem\\u003ec\\u003c/em\\u003e2) generally decreased in bleached corals between T\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e (64% and 47% reduction, respectively), followed by an increase across both bleached and control corals by T\\u003csub\\u003e3\\u003c/sub\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB; Supplemental Fig.\\u0026nbsp;1). Chlorophyll content was very similar between bleached and control corals by T\\u003csub\\u003e4\\u003c/sub\\u003e for chl \\u003cem\\u003ea\\u003c/em\\u003e and T\\u003csub\\u003e5\\u003c/sub\\u003e for chl \\u003cem\\u003ec\\u003c/em\\u003e2.\\u003c/p\\u003e\\u003cp\\u003eSymbiont densities also varied significantly by time and by treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, Supplemental Table\\u0026nbsp;1). Symbiont counts decreased at T\\u003csub\\u003e2\\u003c/sub\\u003e for bleached corals. By T\\u003csub\\u003e4\\u003c/sub\\u003e, bleached and control symbiont counts were about equal and remained similar for the duration of the experiment. Generally, corals maintained the same genus of symbiont throughout the time series (Supplemental Fig.\\u0026nbsp;2). Seven genets were dominated by \\u003cem\\u003eDurusdinium\\u003c/em\\u003e at all time points measured; three genets had \\u003cem\\u003eCladocopium\\u003c/em\\u003e as the dominant symbiont for all time points. Two genets went from being \\u003cem\\u003eCladocopium-\\u003c/em\\u003e to \\u003cem\\u003eDurusdinium-\\u003c/em\\u003edominated between T\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e, regardless of bleaching treatment. One genet changed from \\u003cem\\u003eCladocopium-\\u003c/em\\u003edominance at T\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e to \\u003cem\\u003eDurusdinium-\\u003c/em\\u003edominance at T\\u003csub\\u003e6\\u003c/sub\\u003e, regardless of bleaching status (Supplemental Fig.\\u0026nbsp;2).\\u003c/p\\u003e \\u003cp\\u003eLipid biomass was significantly impacted by time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, Supplemental Table\\u0026nbsp;1). Lipid biomass decreased dramatically in experimentally bleached coral between T\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e but attained the same levels as paired controls by T\\u003csub\\u003e4\\u003c/sub\\u003e and surpassed lipid biomass in control corals at T\\u003csub\\u003e5\\u003c/sub\\u003e and T\\u003csub\\u003e6\\u003c/sub\\u003e. Lipid biomass decreased in control corals around the time of spawning, between T\\u003csub\\u003e5\\u003c/sub\\u003e and T\\u003csub\\u003e6\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003cp\\u003eProteomics\\u003c/p\\u003e \\u003cp\\u003eWe identified 4346 proteins in the \\u003cem\\u003eM. capitata\\u003c/em\\u003e adult proteome across all time points (Supplemental Table\\u0026nbsp;2). In the results that follow, findings from the hierarchical clustering (Supplemental Fig.\\u0026nbsp;3) and the PLS-DA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) are presented together. Hierarchical clustering results are referred to by cluster ID (e.g., NB17\\u0026thinsp;=\\u0026thinsp;cluster #17 from non-bleached controls); PLS-DA protein trends are referred to by the figure panel. Supplemental Tables\\u0026nbsp;2 and 3 contain details on which specific proteins were important for each analysis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eProgression through gametogenesis of non-bleached control colonies\\u003c/p\\u003e \\u003cp\\u003eEach time point in this dataset represents the proteomic profile underlying an important step in the gametogenic process. These processes are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e for the control cohort. In \\u003cem\\u003eM. capitata\\u003c/em\\u003e in Kāneʻohe Bay, the new gametogenic cycle begins between July and October [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD), which is represented by T\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e (September and October) in this study. PLS-DA trends B and C include proteins that increase and decrease, respectively, across early gametogenesis. This time period is marked by increasing abundance of proteins involved in lipid, protein, and carbohydrate metabolism. Many of these general metabolic themes continue through December (T\\u003csub\\u003e3\\u003c/sub\\u003e), which is still within early gametogenesis for \\u003cem\\u003eM. capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSummary of proteomic changes in control corals throughout gametogenesis. All processes and GO terms are elevated at the indicated time point relative to other time points. These results are summaries of the hierarchical clustering and the PLS-DA, summarized in Supplemental Fig.\\u0026nbsp;3, Supplemental Tables\\u0026nbsp;2 and 3, and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTime Point\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eEnriched GO Terms\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eImportant Processes Represented in Proteome\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e1\\u003c/sub\\u003e and T\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;structural constituent of cytoskeleton\\u0026rdquo;; \\u0026ldquo;protein phosphorylation\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eLipid oxidation and metabolism; lipid binding and transport; protein turnover; immune response; carbohydrate metabolism\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e3\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;single-stranded RNA binding\\u0026rdquo;; \\u0026ldquo;semaphorin receptor binding\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCitric acid cycle; protein degradation; amino acid and protein synthesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e4\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen\\u0026rdquo;; \\u0026ldquo;cell adhesion\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eExtracellular matrix remodeling; skeletal mineralization; pentose phosphate pathway; microtubule organizing; transcriptional activation; steroid synthesis; protein degradation; lipid metabolism and transport; mucus\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e5\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;ubiquitin protein ligase activity\\u0026rdquo;; \\u0026ldquo;membrane protein complex\\u0026rdquo;; \\u0026ldquo;dynein complex\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSkeletogenesis; cytoskeleton\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e6\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;microtubule-based movement\\u0026rdquo;; \\u0026ldquo;protein kinase C-activating G protein-coupled receptor signaling pathway\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCytoskeletal structure and organization; sex hormone metabolism; apoptosis; lipid metabolism\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the spring, \\u003cem\\u003eM. capitata\\u003c/em\\u003e undergo a rapid increase in oocyte size, concomitant with an increase in solar radiation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e)[\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. March (T\\u003csub\\u003e4\\u003c/sub\\u003e) also marks a distinctive proteome change in control corals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD and E). Changes in protein abundance span a diverse range of processes including extracellular matrix remodeling, transcriptional activation, and steroid synthesis.\\u003c/p\\u003e \\u003cp\\u003eSpawning in \\u003cem\\u003eM. capitata\\u003c/em\\u003e occurs in June and July and T\\u003csub\\u003e5\\u003c/sub\\u003e and T\\u003csub\\u003e6\\u003c/sub\\u003e capture time periods just days before the first and second spawning dates. Similar to T\\u003csub\\u003e4\\u003c/sub\\u003e, T\\u003csub\\u003e5\\u003c/sub\\u003e represents another inflection point in protein abundance, representing large changes in proteomic profile from T\\u003csub\\u003e4\\u003c/sub\\u003e to T\\u003csub\\u003e5\\u003c/sub\\u003e and from T\\u003csub\\u003e5\\u003c/sub\\u003e to T\\u003csub\\u003e6\\u003c/sub\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD \\u0026amp; E).\\u003c/p\\u003e \\u003cp\\u003eBleaching response and gametogenesis in experimentally bleached colonies\\u003c/p\\u003e \\u003cp\\u003eBleached proteomes had strikingly different temporal trends than those seen in non-bleached controls. Trends of proteomic divergence continued from bleaching through spawning with some results summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. In the hierarchical clusters at the September (T\\u003csub\\u003e1\\u003c/sub\\u003e) time point, there is limited overlap in the proteins that peak and are suppressed at later time points between bleached and control corals (Supplemental Fig.\\u0026nbsp;3). October (T\\u003csub\\u003e2\\u003c/sub\\u003e) represents a large proteomic shift in bleached corals since it is the time point that immediately followed thermal bleaching (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG-J). There is a dramatic change in all of the PLS-DA trends in bleached corals between T\\u003csub\\u003e2\\u003c/sub\\u003e and T\\u003csub\\u003e3\\u003c/sub\\u003e. From the hierarchical clustering, almost twice as many proteins in the bleached colonies increased in abundance at T\\u003csub\\u003e2\\u003c/sub\\u003e (n\\u0026thinsp;=\\u0026thinsp;248) compared to the control colonies (n\\u0026thinsp;=\\u0026thinsp;130), suggesting that T\\u003csub\\u003e2\\u003c/sub\\u003e is a critical time of recovery, supported by proteome remodeling in response to thermal stress (Supplemental Fig.\\u0026nbsp;3). Elevated proteins at T\\u003csub\\u003e2\\u003c/sub\\u003e include some that may be biomarkers of catabolism of stored lipids since the corals remained in tanks with filtered seawater and had limited access to food via heterotrophy.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSummary of proteomic changes in bleached corals throughout bleaching recovery and gametogenesis. All processes and GO terms are elevated at the indicated time point relative to other time points. These results are summaries of the hierarchical clustering and the PLS-DA, summarized in Supplemental Fig.\\u0026nbsp;3, Supplemental Tables\\u0026nbsp;2 and 3, and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTime Point\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eEnriched GO Terms\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eImportant Processes Represented in Proteome\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e1\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;extracellular region\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRetinol dehydrogenase; blood coagulation; immune response\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;membrane\\u0026rdquo;; \\u0026ldquo;signal transduction\\u0026rdquo;; \\u0026ldquo;methionine biosynthetic process\\u0026rdquo;; \\u0026ldquo;glucoronosyltransferase activity\\u0026rdquo;; \\u0026ldquo;regulation of hydrolase activity\\u0026rdquo;; \\u0026ldquo;response to oxidative stress\\u0026rdquo;; \\u0026ldquo;cellular oxidant detoxification\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eLipid catabolism; cellular stress\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e3\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eATP production; extracellular matrix remodeling; lipid uptake; peptide and protein breakdown\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e4\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;histone binding\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eMucus; lipid uptake; gametogenesis\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e5\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026ldquo;carbohydrate derivative transport\\u0026rdquo;; \\u0026ldquo;RNA phosphodiester bond hydroylsis endonucleolytic\\u0026rdquo;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCeramide cycle\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003e6\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eLipid metabolism; cellular stress response; energy transfer to tissues\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eUnlike their paired controls, the bleached corals do not have a dominant proteome signal in March (T\\u003csub\\u003e4\\u003c/sub\\u003e) in the PLS-DA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), the time of rapid egg size increase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). However, T\\u003csub\\u003e4\\u003c/sub\\u003e represents an attainment of proteomic stasis that is maintained for many proteins through spawning (T\\u003csub\\u003e5\\u003c/sub\\u003e and T\\u003csub\\u003e6\\u003c/sub\\u003e). In July (T\\u003csub\\u003e6\\u003c/sub\\u003e), of the 177 proteins with elevated abundance in previously bleached colonies (Supplemental Fig.\\u0026nbsp;3), only sixteen were also elevated in the controls.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eThe full impact of thermal bleaching on population and ecosystem scales is in part determined by the reproductive success of the corals that survive but have still experienced sub-lethal impacts of the stress. Thermal bleaching responses can be variable even within a single species from the same reef [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e], suggesting that individual variability in responses can have reef-wide consequences. We followed a cohort of \\u003cem\\u003eM. capitata\\u003c/em\\u003e through 10 months of recovery after a simulated thermal bleaching event and found that the corals surviving bleaching regain lipid reserves within two months post-bleaching and reacquire symbionts within six months, before spawning occurs. However, differences in oocyte size and protein biomarkers of certain metabolic pathways suggest that even 10 months after thermal bleaching stress, coral still express a phenotype at least partially dictated by prior thermal stress. Although natural bleaching susceptibility in \\u003cem\\u003eM. capitata\\u003c/em\\u003e can be explained by differences in their algal symbionts [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], symbiont type was not strongly associated with the ability of coral colonies to recover from bleaching in this experiment. The focus on the impacts of bleaching on the essential process of gametogenesis in this study provides a novel perspective on thermal bleaching recovery that has not been directly addressed in previous studies on long-term impacts of bleaching using molecular markers (e.g., [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]). Reproductive success is a complicated mosaic that is determined by parent physiology and environmental conditions and culminates in larval survival and settlement; this study provides a detailed depiction of the underpinnings of reproductive success after thermal bleaching.\\u003c/p\\u003e \\u003cp\\u003eImmediately following thermal bleaching in September, \\u003cem\\u003eM. capitata\\u003c/em\\u003e colonies lost their symbionts and much of their lipid reserves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA \\u0026amp; \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). It is well established that thermal stress can disrupt the symbiotic relationship between the coral and its dinoflagellate symbionts (Symbiodiniceae) (e.g., [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]) and once the symbionts are expelled and/or digested, the host loses an important metabolic resource. Symbiont genus did not impact bleaching recovery in this study, as has been observed in natural, field-based bleaching studies (i.e. [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]). We hypothesize that symbiont genus had no impact on bleaching recovery because we intentionally induced complete bleaching in corals hosting varying symbiont community types to generate our bleached cohort, but additional work is needed to distinguish the relationship between thermal resistance and recovery dynamics.\\u003c/p\\u003e \\u003cp\\u003eThere are proteomic signals detected in this study of symbiont rejection and reacquisition in the corals that underwent experimental bleaching. The GS/GOGAT (glutamine synthase/glutamine oxoglutarate aminotransferase) system is the dominant means of N acquisition in a healthy holobiont [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]; this system breaks down when the symbiotic relationship is disrupted and is in part replaced by host urease expression to acquire N [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. In October (T\\u003csub\\u003e2\\u003c/sub\\u003e), there was a decrease in abundance of coral glutamine synthetase (part of the GS/GOGAT system) and an increase in urease enzymes relative to control corals, suggesting a shift in host N acquisition reflecting the loss of photosynthate (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eCoral N acquisition via symbiont pathways is still suppressed in December (T\\u003csub\\u003e3\\u003c/sub\\u003e), reinforcing that symbionts are not yet providing all the resources the host needs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Isocitrate dehydrogenase is another enzyme associated with GS-GOGAT and may be a biomarker of symbiotic dysfunction; some homologs of this enzyme are elevated in bleached compared to control colonies in December (T\\u003csub\\u003e3\\u003c/sub\\u003e) and March (T\\u003csub\\u003e4\\u003c/sub\\u003e) (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Thus, during this critical period of early gametogenesis, the previously bleached corals are relying predominantly on heterotrophic feeding to rebuild lipid reserves.\\u003c/p\\u003e \\u003cp\\u003eSymbiont reacquisition was almost complete by March (T\\u003csub\\u003e4\\u003c/sub\\u003e) in previously bleached coral and abundances are about equal with their non-bleached paired controls. In the proteome, there was an almost eight-fold decrease in Rab11a abundance in March (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), a protein that is suppressed during healthy symbiosis [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Healthy, photosynthesizing symbionts would provide the coral host metabolic by-products, such as simple carbohydrates, which the host then metabolizes [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Two metabolic enzymes (pyranose oxidase and succinate-semialdehyde dehydrogenase), which may be involved in processing of symbiont-derived resources, decrease post-bleaching and rebound through symbiont reacquisition, perhaps signaling a reestablishment of symbiont byproduct-derived carbohydrate metabolism (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eDuring physiological stress, organisms may reallocate energetic resources to respond to the stress while maintaining essential physiological functions. In corals, skeletogenesis decreases immediately following bleaching stress or reduction in photosynthate (e.g., [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]). In the \\u003cem\\u003eM. capitata\\u003c/em\\u003e proteome of experimentally bleached corals, we see a suppression in the abundance of all isoforms of carbonic anhydrase, a skeletogenesis protein, suggesting a decrease in skeletal growth relative to non-bleached controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), which has been previously observed [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. This trend in carbonic anhydrase abundance is recapitulated in the \\u003cem\\u003eM. capitata\\u003c/em\\u003e transcriptome post-thermal stress [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. Another aspect of the coral response to bleaching is an increase in reactive oxygen species (ROS) and a concomitant increase in ROS scavengers and repair proteins to mitigate and respond to cellular damage [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. As in [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], homologs of glutathione-S-transferase and caspase were elevated in bleached corals compared to non-bleached controls in October, suggestive of a ROS response (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). ROS response proteins elevated in October (T\\u003csub\\u003e2\\u003c/sub\\u003e) also include peroxidasin (a known inhibitor of ROS), as well as proteins that are likely responding to the consequences of cellular damage: protein disulfide isomerase A6 (inhibits aggregation of misfolded proteins) and leukocyte elastase inhibitor (protects cells from proteases released into the cytoplasm during stress). These proteins all potentially represent a cellular effort to repair and mitigate damage to DNA, proteins, and other cellular structures in the aftermath of thermal stress.\\u003c/p\\u003e \\u003cp\\u003eThe timing of symbiont loss, which corresponds to when most natural bleaching events occur, overlaps with the onset of gametogenesis in \\u003cem\\u003eM. capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Gametogenesis is an energy intensive process, and the combined effect of lipid store catabolism with the loss of symbiont resources could conceivably deplete the energy store necessary to form viable gametes. October (T\\u003csub\\u003e2\\u003c/sub\\u003e) also represents the start of a months-long recovery period for bleached corals. During these months post-bleaching, we show evidence that bleached coral survive bleaching by first catabolizing lipid reserves (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) and then switching to heterotrophic feeding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) to rapidly restore lipid levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC) while reacquiring symbionts (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA) and acquire enough autotrophically derived C before spawning (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) to successfully undergo gametogenesis, with potential tradeoffs in egg size (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003eLipid and proteomic data in the present study suggest that bleached corals began to catabolize their lipid reserves post-bleaching to make up for the loss of metabolic products from their symbionts. The rapid mobilization of stored lipids to compensate for reduced photosynthate after bleaching has been well documented in \\u003cem\\u003eM. capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and other coral species [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Many protein biomarkers detected at this time point also confirm a probable up-regulation of lipid catabolism immediately post-bleaching (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e and Supplemental Fig.\\u0026nbsp;4).\\u003c/p\\u003e \\u003cp\\u003eLipids are essential macromolecules in the development of gametes in marine organisms (e.g., [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]) and catabolism of lipids for other physiological purposes may have lasting impacts on the gametogenic process. The experimentally bleached corals depleted lipid reserves through the first two months post-bleaching (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC), with proteomics revealing some of the molecular mechanisms of lipid catabolism during this time. Phospholipase B is one of the proteins that follows a trend of elevated abundance in bleached coral compared to non-bleached controls in October (T\\u003csub\\u003e2\\u003c/sub\\u003e) [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e; Supplemental Fig.\\u0026nbsp;4). Even if symbionts remain in the bleached corals, thermally stressed symbionts may provide fewer lipids to their host [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Therefore, the lipid metabolism enzymes that are elevated at this time point are either acting on 1) the few lipids provided by the remaining symbionts, 2) heterotrophically-derived lipids, or 3) storage lipids from the coral tissue. \\u003cem\\u003eM. capitata\\u003c/em\\u003e use stored lipid reserves when photosynthate and heterotrophy are unavailable [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], while as much as 70% of \\u003cem\\u003eM. capitata\\u003c/em\\u003e lipids can be heterotrophically derived immediately post-bleaching [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. In October, the corals had not yet been outplanted to racks on the reef and therefore had access to only limited suspended plankton in tanks for heterotrophy. Given the low number of symbionts and rapid decrease in lipid reserves, the corals likely derived energy from their stored lipids at T\\u003csub\\u003e2\\u003c/sub\\u003e. Lipids are the main energy source that corals allocate to their gametes [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e] and the depletion of these stores in early gametogenesis impacts gamete production.\\u003c/p\\u003e \\u003cp\\u003eIn December, bleached corals had not yet reacquired all their symbionts, yet lipid content was almost on par with their non-bleached control counterparts. This rapid reacquisition of lipids has been previously observed in \\u003cem\\u003eM. capitata\\u003c/em\\u003e and may be in part attributable to more efficient resource conservation due to a lower respiration rate than other coral species [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], as well as heterotrophic feeding. In December, many lipid hydrolysis enzymes, potentially linked to food acquisition and lipid digestion, were at relatively high abundance in bleached coral compared to non-bleached controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Some proteins involved in peptide and protein breakdown were also elevated, and two proteins involved in ATP synthesis increased in abundance from October (T\\u003csub\\u003e2\\u003c/sub\\u003e) through March (T\\u003csub\\u003e4\\u003c/sub\\u003e, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Combining all the molecular evidence, it suggests that \\u003cem\\u003eM. capitata\\u003c/em\\u003e engages in external food acquisition and breakdown, as well as cellular energy production despite low levels of symbionts. \\u003cem\\u003eM. capitata\\u003c/em\\u003e its known for its trophic plasticity and can acquire the food necessary to fulfill its daily metabolic needs with heterotrophy alone [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. This flexibility in the nutrient acquisition is not present across all coral species [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e] and may be part of the phenotype of increased bleaching resilience observed in \\u003cem\\u003eM. capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Between October (T\\u003csub\\u003e2\\u003c/sub\\u003e) and December (T\\u003csub\\u003e3\\u003c/sub\\u003e), bleached \\u003cem\\u003eM. capitata\\u003c/em\\u003e are likely relying on heterotrophy almost exclusively to rebuild lipid reserves, which could provide energy to support spawning and/or survival during a future bleaching event.\\u003c/p\\u003e \\u003cp\\u003eMarch (T\\u003csub\\u003e4\\u003c/sub\\u003e) represents a peak in lipid biomass for these corals since lipids are allocated to oocytes, and adult tissue lipid levels decline post-spawning [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. This period of lipid accumulation leading up to allocation to oocytes is essential for embryo and larval development and survival in their planktonic phase [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Some lipid metabolism enzymes were relatively lower in bleached corals in March (e.g., lipase, phospholipase D3, saposin domain-containing protein), while others were relatively elevated (e.g., phospholipase A2 and B, prosaposin). Two hypotheses for these trends could be that 1) the bleached coral have \\\"overcompensated\\\" for the depletion of lipid reserves during bleaching and have upregulated their lipid metabolism and storage pathways via increased heterotrophy and/or the return of symbiosis coinciding with increased light and temperature during spring months. Alternatively, 2) bleached coral may be dedicating relatively less lipid to their oocytes and thus maintaining a higher reserve in their somatic tissue than controls. The elevated level of lipids in adult coral is likely due to \\u003cem\\u003eM. capitata\\u003c/em\\u003e's ability to store heterotrophically derived carbon, as suggested by [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e], and as evidenced by the trends seen in early lipid reacquisition in this study.\\u003c/p\\u003e \\u003cp\\u003eThe June (T\\u003csub\\u003e5\\u003c/sub\\u003e) and July (T\\u003csub\\u003e6\\u003c/sub\\u003e) time points represent the time periods just before spawning events and are marked in control coral by a decrease in lipid biomass (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC) and another inflection point in trends of protein abundance in the PLS-DA trends (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD \\u0026amp; \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). The dip in lipid biomass is not observed in bleached corals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC); higher lipid levels may help these corals survive future bleaching events [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. The dip in lipid levels of adult tissues is a consistent trend across broadcast-spawning corals that marks the allocation of lipids to oocytes [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. It has been hypothesized that the drop in tissue lipid content with spawning is directly correlated with energy allocation to gametes [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Even though bleached \\u003cem\\u003eM. capitata\\u003c/em\\u003e prioritize energy transfer to oocytes [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], the lack of early access to autotrophic C may have resulted in less energy allocation to gametes in bleached corals, compared to controls. In the proteome, homologs of vitellogenin, a major egg yolk protein, are all at lower abundance in previously bleached coral compared to non-bleached controls in June, suggesting less lipid allocation to oocytes (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eRepeated bleaching events can lead to increased mortality in some species, perhaps because energetic reserves used to survive prior stressful events are depleted [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. \\u003cem\\u003eM. capitata\\u003c/em\\u003e in the dynamic Kāneʻohe Bay may have evolved physiological mechanisms to survive repeated environmental stresses (e.g. [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]). The tradeoff, however, could have implications for larval viability if there are less maternal lipids to sustain the non-feeding stages of development and to maintain positive buoyancy [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eWhile the dominant protein abundance trends in bleached corals were in response to bleaching, a significant proteomic shift occurred in non-bleached controls during March (T\\u003csub\\u003e4\\u003c/sub\\u003e), coinciding with the period of most rapid oocyte size increase and onset of spermatogenesis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE \\u0026amp; \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eF). A wide range of functions are represented by the proteins of control corals that peak in abundance in March. The proteins encompass functions in DNA and cellular replication, protein turnover, and signaling via neurons and neuropeptides (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Signals of extracellular matrix reorganization, a basal process essential to gametogenesis [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], were detected in the control corals: two proteins that negatively regulate or degrade the ECM were at decreased abundance in March relative to previous time points (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). These proteins represent the complex physiological processes necessary to support gametogenesis.\\u003c/p\\u003e \\u003cp\\u003eAs in other animals, sex hormones regulate reproductive maturation in corals (e.g., [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]) and we identified four proteins with likely sex hormone regulation functions that are impacted by thermal bleaching. The hierarchical clusters that peak in March (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) include 1) steroid 17-alpha-hydroxylase (also known as CYP17A1), a protein important in regulation of sex hormones during gametogenesis [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e] (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). CYP17A1 abundance is not impacted by bleaching recovery at the time points investigated. 2) Steroidogenic acute regulatory protein (StAR) abundance is depressed in previously bleached corals at T\\u003csub\\u003e2\\u003c/sub\\u003e and T\\u003csub\\u003e3\\u003c/sub\\u003e and then increases to levels higher than those in controls at T\\u003csub\\u003e5\\u003c/sub\\u003e. StAR is an essential early step in steroid synthesis and has been identified in other corals genomes [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e] (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). 3) Protein flightless-1 and 4) 17B hydroxysteroid dehydrogenase are both at relatively low abundance in bleached corals at T\\u003csub\\u003e4\\u003c/sub\\u003e. The former may regulate transcription downstream of estrogen and androgen receptors, and the latter is known to regulate estrogen and androgen levels in mammals and has known estrogen activity in corals [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e] (Supp. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). In the weeks leading up to spawning, sex hormones increase in \\u003cem\\u003eM. capitata\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e] and the proteins regulating these hormones detected in this study give further insight into patterns of sex hormone production during gametogenesis.\\u003c/p\\u003e \\u003cp\\u003eIn June (T\\u003csub\\u003e5\\u003c/sub\\u003e), oocyte and spermatocyte stages were similar between bleached and control coral, but oocyte Feret diameter was significantly smaller in previously bleached corals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Oocyte diameter reduction is a common effect of bleaching across coral species [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Even though bleached coral had attained the same lipid levels as controls by December, these lipid stores are likely derived mostly from heterotrophically derived carbon since symbiont levels were still low. \\u003cem\\u003eM. capitata\\u003c/em\\u003e colonies do not allocate heterotrophically derived carbon to oocytes, only autotrophic carbon [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Previously bleached corals likely had less autotrophically derived C for their oocytes than controls. \\u003cem\\u003eM. capitata\\u003c/em\\u003e and other corals provision eggs with lipids, proteins, symbionts, and photoprotectant molecules [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]; adult bleaching can reduce the amounts of these molecules allocated to oocytes [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e], which may be reflected in the smaller size of bleached corals\\u0026rsquo; oocytes in this study.\\u003c/p\\u003e \\u003cp\\u003eBleaching impacts on coral gametogenesis vary both across species and populations within a species. If a coral population is highly tolerant to bleaching stress, they may still be able to execute full gametogenesis as seen in this study and others (e.g. [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]). Bleaching tolerance may be species-specific or genetically determined but may also be a result of acclimatization over multiple bleaching events (e.g., [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]). The severity of the bleaching event and/or a history of thermal bleaching exposure can dictate whether and to what degree a coral can achieve gametogenesis [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. \\u003cem\\u003eM. capitata\\u003c/em\\u003e in Kāneʻohe Bay experienced thermal stress and may have bleached in 2014 and 2015 (two and three years before this study, respectively; [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]) and seem to have evolved resilience to disturbances [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Their combined genetic and ecological histories may give them the physiological capacity to accomplish gametogenesis despite bleaching, while other species or populations may fail. Some of the functional protein biomarkers identified here may help identify essential traits and mechanisms of thermal bleaching-resilient phenotypes.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eM. capitata\\u003c/em\\u003e demonstrated a potential for resilience to thermal bleaching stress as well as an ability to rapidly recover symbionts and lipid levels post-bleaching. However, the bleached colonies did not have access to symbiont-provided autotrophic carbon during the critical months of early gametogenesis, which likely resulted in a time lag in upregulating some of the physiological processes necessary for gametogenesis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). One result of this delay in resource access was significantly smaller oocytes in bleached corals. Corals in Kāneʻohe Bay have experienced increasingly frequent thermal bleaching events in recent years, the effects of which may be seen in smaller oocytes across the population when compared with oocyte sizes from previous years (e.g., [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e], [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]). Additionally, previous work has suggested that the impacts of bleaching can be long-lasting and more detrimental for coral sperm than for eggs [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. A longer-term study that follows these corals across years and multiple bleaching events would clarify whether these strategies facilitate faster recovery from bleaching events. Additionally, it is important to better understand if there are any short- or long-term population-level consequences in allocating less of the adult lipid biomass to gametes in terms of the number of viable embryos and successfully settled juveniles. Since both adult coral survival and juvenile settlement success are integral to continued reef survival, it is not enough that we understand \\u003cem\\u003eif\\u003c/em\\u003e corals survive thermal bleaching; we also must understand the cost of that survival.\\u003c/p\\u003e\"},{\"header\":\"METHODS\",\"content\":\"\\u003cp\\u003eCoral Selection and Bleaching\\u003c/p\\u003e \\u003cp\\u003eDetails for coral collection and historical bleaching regime can be found in [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Seventy-four \\u003cem\\u003eM. capitata\\u003c/em\\u003e coral (approximately 30 cm in diameter) were collected from patch reefs located in Kāneʻohe Bay, Oʻahu, Hawaiʻi around the Hawaiʻi Institute of Marine Biology (HIMB, 21.428\\u0026deg;N, 157.792\\u0026deg;W) in August 2017. Each coral was further divided in half so that genetically identical halves (i.e. ramets of a genet) were replicated in the control and treatment conditions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Experimental corals were acclimated in flow-through outdoor tanks (three tanks per treatment group) for 7\\u0026ndash;10 days. In September (T\\u003csub\\u003e1\\u003c/sub\\u003e), the period when bleaching events typically occur in Hawaiʻi [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], corals in the bleaching treatment were exposed to warmer water temperatures (30\\u0026deg;C) to simulate these conditions (Supplemental Fig.\\u0026nbsp;5). Experimental corals were then placed on racks off HIMB after the sampling time point on October 1, 2017 (T\\u003csub\\u003e2\\u003c/sub\\u003e) to monitor their recovery and physiological response to the in situ environment through July (T\\u003csub\\u003e6\\u003c/sub\\u003e) (Supplemental Fig.\\u0026nbsp;6). Mortality and bleaching assessments were conducted weekly using the Coral Watch Coral Health Chart [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe time points when corals were sampled are: 1) at the end of August (August 30, 2017) after corals acclimated in the tanks but before they were bleached (T\\u003csub\\u003e1\\u003c/sub\\u003e); 2) in October, 24 hours after bleached corals were returned to ambient temperature (T\\u003csub\\u003e2\\u003c/sub\\u003e; October 1, 2017); 3) in December (T\\u003csub\\u003e3\\u003c/sub\\u003e; December 20, 2017); 4) in March, during the period of the rapid increase in egg size (T\\u003csub\\u003e4\\u003c/sub\\u003e; March 29, 2018) [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]; 5) in June, at the beginning of the spawning season, when symbionts are vertically transferred to gamete bundles (T\\u003csub\\u003e5\\u003c/sub\\u003e; June 8, 2018) [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]; 6) and in July, later in the spawning season (T\\u003csub\\u003e6\\u003c/sub\\u003e; July 9, 2018). Additional details are provided in Supplemental Methods.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eHistology\\u003c/p\\u003e \\u003cp\\u003eHistology slides were made from serial sections of \\u003cem\\u003eM. capitata\\u003c/em\\u003e tissue samples collected in June 2018 (T\\u003csub\\u003e5\\u003c/sub\\u003e). Slides were stained with hematoxylin and eosin before observation under a compound microscope, photographed using a camera attachment (model DS-Fi3, Nikon Instruments Inc.), and analyzed with NIS-Elements imaging software. Oocyte measurements were made in Image-J software version 1.52 using Feret\\u0026rsquo;s Statistical Diameter to estimate size. Only oocytes with visible nuclei were measured to ensure standardization of measurements along the widest axis of the oocyte. Developmental stage of gametes was assessed using morphological guidelines and Feret diameter size ranges [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAlthough the response variable, Feret diameter, was not normally distributed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA), the sample size was sufficiently large (n\\u0026thinsp;=\\u0026thinsp;858) to fulfill assumptions of the Central Limit Theorem that the sampling distribution is approximately normally distributed. Means, standard deviations, and ranges of oocyte Feret diameter were calculated for both control and experimentally bleached coral. Feret differences and oocyte and sperm stages between bleached and control corals were evaluated following [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Additional details are provided in Supplemental Methods.\\u003c/p\\u003e \\u003cp\\u003eChlorophyll, symbiont counts, symbiont clades, and lipids\\u003c/p\\u003e \\u003cp\\u003eChlorophyll \\u003cem\\u003ea\\u003c/em\\u003e (Chl \\u003cem\\u003ea\\u003c/em\\u003e) and dinoflagellate symbiont (Symbiodiniceae) counts were analyzed as reported by [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Briefly, Chl \\u003cem\\u003ea\\u003c/em\\u003e was extracted on ground whole coral (tissue and skeleton) samples using 100% acetone [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e], determining absorbance at 630, 663, and 750 nm, and standardized to total dry tissue weight.\\u003c/p\\u003e \\u003cp\\u003eFor symbiont counts, dinoflagellate symbionts were separated from ground whole coral samples; isolated pellets were resuspended in filtered seawater with 1% formalin and 2\\u0026ndash;3 drops of Lugol\\u0026rsquo;s iodine. Three subsamples were counted using a hemocytometer, and the mean count was reported.\\u003c/p\\u003e \\u003cp\\u003eRelative symbiont abundances (genera \\u003cem\\u003eCladocopium\\u003c/em\\u003e and \\u003cem\\u003eDurusdinium\\u003c/em\\u003e) were measured with qPCR for tissue samples taken in September, October, and July (T\\u003csub\\u003e1\\u003c/sub\\u003e, T\\u003csub\\u003e2\\u003c/sub\\u003e, and T\\u003csub\\u003e6\\u003c/sub\\u003e DNA was extracted from tissue using a CTAB-chloroform protocol (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003edx.doi.org/10.17504/protocols.io.dyq7vv\\u003c/span\\u003e\\u003cspan address=\\\"10.17504/protocols.io.dyq7vv\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cb\\u003e)).\\u003c/b\\u003e Proportions of \\u003cem\\u003eCladocopium\\u003c/em\\u003e and \\u003cem\\u003eDurusdinium\\u003c/em\\u003e symbiont cells in each sample were quantified with qPCR using actin assays [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e] on an Agilent AriaMX system with two technical replicates run for 40 cycles. Additional details are provided in Supplemental Methods.\\u003c/p\\u003e \\u003cp\\u003eLipids were extracted from ground whole coral samples with a 2:1 chloroform: methanol solution, 0.88% KCl and 100% chloroform washes [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. Extracted lipid samples were dried to a constant weight under grade 5.0 N\\u003csub\\u003e2\\u003c/sub\\u003e gas and standardized to total dry tissue weight.\\u003c/p\\u003e \\u003cp\\u003eLinear mixed effects models (lmer in the lme4 package [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]) were applied to determine significant differences in chlorophyll \\u003cem\\u003ea\\u003c/em\\u003e and \\u003cem\\u003ec2\\u003c/em\\u003e concentration, symbiont counts, and total lipids with bleaching status and time as fixed effects and coral genet as a random effect. Significant effects (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) were determined in lmerTest [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. There was not enough data for the symbiont genera to fit a generalized linear model, but qualitative results are described.\\u003c/p\\u003e \\u003cp\\u003eProteomics\\u003c/p\\u003e \\u003cp\\u003eProtein digestions were conducted with 100 \\u0026micro;g of protein per coral sample as described in [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. \\u003cem\\u003eM. capitata\\u003c/em\\u003e samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC‒MS/MS) on a Q‒Exactive‒HF (Thermo) in Data Dependent Acquisition (DDA) mode.\\u003c/p\\u003e \\u003cp\\u003eFrom each mass spectrometry experiment, \\u003cem\\u003eM. capitata\\u003c/em\\u003e peptides were identified and proteins were inferred using a proteome derived from the \\u003cem\\u003eM. capitata\\u003c/em\\u003e genome (NCBI Bioproject Accession no. PRJNA509219) [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. The \\u003cem\\u003eM. capitata\\u003c/em\\u003e proteome was concatenated with a predicted proteome for Symbiodiniaceae genus \\u003cem\\u003eCladocopium\\u003c/em\\u003e (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://symbs.reefgenomics.org/download/\\u003c/span\\u003e\\u003cspan address=\\\"http://symbs.reefgenomics.org/download/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e)\\u003c/span\\u003e and 50 common contaminants (cRAPome [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]); the final proteome database contained 99,221 sequences. The raw MS data (PRIDE accession PXD021262, reviewer username\\u0026thinsp;=\\u0026thinsp;\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003ereviewer93258@ebi.ac.uk\\u003c/span\\u003e, password\\u0026thinsp;=\\u0026thinsp;r5h4H1vo) were searched against the protein database using Comet v 2019.01 rev.5 [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Concatenated target\\u0026ndash;decoy database searches were followed by PeptideProphet and ProteinProphet [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e] with a probability cut-off of 0 to allow for FDR cut-off downstream in Abacus. Resulting data files across all samples were analyzed with Abacus [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e] to generate consistent protein inferences across replicates and to calculate normalized spectral abundance factors (NSAF) with an FDR cut-off of 0.01 (protein probability of 0.91). Proteins were included in downstream analyses if two unique peptides were identified across all mass spectrometry experiments and if they were not flagged as outliers [\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eProteins important to experimental bleaching response and/or to gametogenesis were identified with two main methods described below: 1) hierarchical clustering and 2) discriminant analysis by partial least squares (PLS-DA). These two methods are complementary and were used to identify which molecular processes change over time and how those changes may impact bleaching recovery and the timeline of gametogenesis. Additional proteomics details are provided in Supplemental Methods.\\u003c/p\\u003e \\u003cp\\u003eAll analyses described above were accomplished in R [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. All data files and R code used for analyses are available on Dryad: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://datadryad.org/stash/share/TH40Lri9NsCmVVGpYe92iJ2jTfVsvJT0ryORkPSba70\\u003c/span\\u003e\\u003cspan address=\\\"https://datadryad.org/stash/share/TH40Lri9NsCmVVGpYe92iJ2jTfVsvJT0ryORkPSba70\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eETS performed the protein extractions, data analysis and interpretation, and was the primary author of the manuscript. ED contributed to data analysis and interpretation. TB was responsible for all field experiments and assisted with lab work. JBA assisted with field work and symbiont counts. CHB did the symbiont counts. MR contributed to data analysis. JD and CDK performed the qPCR of the symbiont genera. LJR contributed to funding acquisition, study design, field experiments, and performed the lipid analyses. BLN contributed to the study design, protein extractions, and performed the mass spectrometry analysis. JLPG conceived of the experimental time-series study design and led the field work and project management. All co-authors contributed to writing of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eThis research was supported by the National Science Foundation\\u0026rsquo;s Division of Integrative Organismal Systems, Integrative Ecological Physiology Program (NSF IOS-IEP) 1655682 to JP-G and BN, NSF IOS-IEP 1655888 to LJR and NSF CAREER (BIO-OCE, 2044840) and the Sloan Foundation Fellowship to JP-G, and start-up funding from the University of Southern California to CDK. Additional support came from the University of Washington's Proteomics Resource (UWPR95794). We thank Dr. Ruth Gates and the Gates Coral Laboratory for our sponsorship at the Hawai\\u0026rsquo;i Institute of Marine Biology. We thank G. Kreitman, M. Jaffe, S. Frangos, J. Davidson, and E. Lenz, for field and laboratory support. We would also like to acknowledge the essential work done by the Genome Sciences IT group to support all our work. ETS would like to thank IJE and EGE for continuing inspiration.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe raw proteomics mass spectrometry files are available on PRIDE under accession PXD021262, reviewer username = reviewer93258@ebi.ac.uk, password = r5h4H1voAll R code and files necessary for the statistical analyses and plots presented in this manuscript are available on Dryad: https://datadryad.org/stash/share/TH40Lri9NsCmVVGpYe92iJ2jTfVsvJT0ryORkPSba70\\u003c/p\\u003e\\u003cp\\u003eCOMPETING INTERESTS\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no competing interests to declare.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eHughes, T.P., Baird, A.H., Bellwood, D.R., Card, M., Connolly, S.R., Folke, C., Grosberg, R., Hoegh-Guldberg, O., Jackson, J.B.C., Kleypas, J., et al. Climate change, human impacts, and the resilience of coral reefs. 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R package version 2.6-4, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e\\u0026lt;https://CRAN.R-project.org/package=vegan\\u0026gt;\\u003c/span\\u003e\\u003cspan address=\\\"http://%3Chttps://CRAN.R-project.org/package=vegan%3E\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e (2020).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"thermal stress, reproduction, histology, reef-builder, proteomics\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4559943/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4559943/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThermal bleaching, or the loss of symbiotic algae that provide most energetic resources for the coral host, is an increasing threat to reefs worldwide and is projected to worsen with climate change. While bleaching is a well-recognized threat, the impact on the process of reproduction in bleaching survivors is not well resolved, despite being central to coral resilience. \\u003cem\\u003eMontipora capitata\\u003c/em\\u003e can survive bleaching while completing a full gametogenic cycle, offering an ideal system to study gametogenic resilience and physiological tradeoffs. We experimentally bleached fragments of \\u003cem\\u003eM. capitata\\u003c/em\\u003e colonies and followed their gametogenesis and physiological responses for 10 months (six time points). All bleached colonies produced gametes at the same time as controls, suggesting that reproductive processes were energetically prioritized. However, proteomic analysis revealed tradeoffs and delays in activating key physiological processes earlier in gametogenesis in areas such as skeletal growth and reproductive hormone synthesis. Tradeoffs during the gametogenic cycle, likely a direct response to thermal bleaching, resulted in smaller oocytes from bleached colonies, potentially indicating decreased transfer of parental resources to gametes. While gametogenesis is likely to continue in this species, it is unknown how the viability and success of future offspring may be impacted by future bleaching events.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Reproductive Resilience: Pathways to Gametogenic Success in Montipora capitata after Bleaching \",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-07-12 05:40:17\",\"doi\":\"10.21203/rs.3.rs-4559943/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-08-01T18:06:48+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-22T11:32:42+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-21T15:37:03+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-18T05:56:13+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"112740513135504820123909352924237170199\",\"date\":\"2024-07-07T07:10:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"276180486094090685052362170502786733606\",\"date\":\"2024-07-07T04:45:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"123354160481243942495659664388336759016\",\"date\":\"2024-07-06T23:24:54+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"129579020420578555066272180559786652948\",\"date\":\"2024-07-05T00:24:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-07-04T22:11:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-07-01T18:54:37+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-06-26T12:30:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-06-22T04:41:10+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2024-06-10T19:59:58+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f2bf3755-ad48-4f9f-8de1-a7912bed4fb9\",\"owner\":[],\"postedDate\":\"July 12th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":34225517,\"name\":\"Biological sciences/Ecology/Climate change ecology\"},{\"id\":34225518,\"name\":\"Biological sciences/Molecular biology/Proteomics\"}],\"tags\":[],\"updatedAt\":\"2024-11-18T19:25:59+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4559943\",\"link\":\"https://doi.org/10.1038/s41598-024-78768-7\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2024-11-13 15:58:01\",\"publishedOnDateReadable\":\"November 13th, 2024\"},\"versionCreatedAt\":\"2024-07-12 05:40:17\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-024-78768-7\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-024-78768-7\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4559943\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4559943\",\"identity\":\"rs-4559943\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}