Transgenerational metabolomic signatures of bleaching resistance in corals

doi:10.21203/rs.3.rs-4926721/v1

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Transgenerational metabolomic signatures of bleaching resistance in corals

https://doi.org/10.21203/rs.3.rs-4926721/v1

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Coral bleaching is one of the greatest climate-driven threats to the persistence of tropical reef ecosystems. This necessitates the identification of attributes associated with coral resistance and resilience to thermal stress both within and between generations. Here, we used metabolomics to demonstrate that biochemical signatures associated with heat-induced bleaching of Montipora capitata are passed between generations. There were metabolomic signatures of parental bleaching phenotype in sperm, eggs, embryos, larvae, and subsequent juvenile corals formed by selectively breeding bleaching resistant or susceptible parents. Metabolome source mapping showed that these thermal tolerance signatures were from both the coral host and the algal symbiont and spanned a variety of molecular families. One of the strongest markers of transgenerational heat tolerance was the saturation state of DGCC betaine lipids, a molecular family previously associated with thermal tolerance in dinoflagellate symbionts of corals. Though the saturation state of the DGCC lipids was strongly linked to algal genotypes, which are known to be vertically transmitted in M. capitata, even coral progeny that contained the more thermally susceptible Cladocopium algae showed increased saturation of this lipid group if their parents had resisted recent bleaching events. This work provides evidence for biochemical inheritance as a potential mechanism for transgenerational acclimatization to warming oceans, which has implications for reef restoration and resilience in the face of climate change.

Biological sciences/Ecology/Microbial ecology

Biological sciences/Ecology/Conservation biology

Corals and single-celled algae in the family Symbiodiniaceae form one of the most ancient and ecologically important intracellular symbioses¹. Together, the coral animal, its algal symbiont, and their associated viruses and microbes form the coral holobiont^2,3. Metabolites are exchanged between members of this biological consortium for the ecological and evolutionary benefit of the holobiont as a whole^4,5. For example, the coral provides nitrogenous metabolites in exchange for photosynthetic products from the algae, which can provide up to 95% of the animal’s energetic needs⁶. Algal photosynthate is primarily glycerol with smaller quantities of glucose, lipids, free fatty acids, amino acids, and organic acids^7,8, and serves as a major source of organic carbon for the coral holobiont. This metabolic exchange is the foundation of the coral-dinoflagellate symbiosis, but it can be disrupted by environmental stressors such as increasingly warm oceans⁹. Periods of prolonged heat stress can cause the complete breakdown of this symbiotic partnership by inducing coral bleaching, which can lead to mortality and the loss of function across coral reef ecosystems.

Bleaching, where corals lose their intracellular algal symbionts, is a complex biological process involving genetic regulation and biochemical changes in both the algal symbionts and the coral animal^10–12. Extensive work has shown the transcriptional^13–15, microbial¹⁵, and metabolomic^10–12,16 impacts of heat stress in the coral holobiont. Previous work using untargeted metabolomics has also shown that the bulk metabolome of the holobiont is strongly correlated with thermal tolerance phenotypes, even during periods without thermal stress¹⁰. The use of untargeted metabolomics allows for the assessment of metabolite pools in all members of holobiont simultaneously and provides a robust picture of the physiological status of the coral host, the algal symbiont, and the bacterial microbiome^{10,12,17–19}. These changing metabolites relate to the health status of a colony and can be early signals of a stressed metabolic state under environmental change²⁰. The increasing frequency and severity of coral bleaching events^21–24 necessitates the understanding of the mechanisms involved in the ecology and evolution of thermal tolerance in corals and the identification of biomarkers or other indicators of thermal stress.

Here we used untargeted metabolomic profiling of adults, gametes, embryos, larvae and juveniles from a model population of Montipora capitata with known, reproducible thermal tolerance phenotypes during natural bleaching events^25,26. This trait is highly heritable in larvae and juveniles due to both genetic and non-genetic (e.g., symbiont) factors²⁵, representing a model system for understanding the adaptive potential of coral reefs. Our results demonstrate the transfer of biochemical traits across life history stages, highlighting the importance of parental provisioning of offspring in a reef-building coral and clarifying one mechanism of non-genetic heritability which can support the long-term persistence of corals in the face of climate change.

Metabolomic Variation Through the Coral Life Cycle

We collected samples of adult corals and their sperm and eggs from 47 parent colonies of the reef-building coral M. capitata. These parent colonies represent those with different natural bleaching phenotypes (i.e., some parent colonies bleached during recent thermal stress events, while others did not). After collecting samples of sperm and eggs, the remaining gametes were combined for bulk fertilization into separate pools sourced from exclusively bleached or exclusively non-bleached parents (see methods). Newly formed embryos (12-hours post fertilization), free swimming larvae (15-, 40-, 72-, 90-, and 102-hours post fertilization), and settled juveniles were collected from each pool and all samples were analyzed using untargeted metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The LC-MS/MS data identified 7,532 metabolite features (192 with spectral annotations in the Global Natural Products Social Molecular Networking database, GNPS²⁷ libraires), of which 310 were unique to adults, 394 to eggs, 39 to sperm, 6 to larvae, 8 to juveniles, and none to embryos (a combination of sperm and egg, Fig. S1). Furthermore, 313 metabolite features were found to be ubiquitous across all life history stages. The metabolome of each cell type (adults, egg, sperm, embryo, larvae, juvenile) were found to be significantly different from each other (PERMANOVA, p = 0.001, Fig. 1A), with the aposymbiotic sperm metabolomes distinctly different from the other life stages. Adult corals and eggs also formed distinct groups, with larval samples in between, while embryo samples clustered similarly to the developing larvae and juveniles. The developing larvae showed a progressively changing metabolome profile over 120 hours of development (PERMANOVA, p = 0.001, Fig. 1B). There was substantial differentiation between embryos at the prawn chip stage (~ 15 hours post fertilization, hpf), developing larvae (40–72 hpf), and fully competent swimming larvae (+ 72 hpf). Differences in the metabolome were first assessed using diversity metrics (richness, entropy, and evenness). Adults, eggs, and larvae had the highest richness and alpha-diversity (Shannon entropy) with the sperm samples, embryos, and juveniles having significantly lower metabolite diversity (one-way ANOVA on ranks, p ≤ 0.01; Fig. 1C). The alpha-diversity of developing larvae also increased during development over 120 hours (Fig. 1D). Furthermore, we compared metabolomic distance (pairwise Bray-Curtis dissimilarity) between gametes and their parent colony (parent gamete pairs; PGP) versus gametes and non-parent colonies, demonstrating a significantly smaller metabolomic distance between eggs and their parent colony when compared to non-parent colonies (Student’s T-test, p ≤ 0.01; Fig. 1E). This difference in metabolic distance between parent and non-parent colonies was not observed for sperm (Fig. 1F).

Transgenerational Thermal Tolerance Signatures in the Coral Metabolome

Permutational multivariate analysis of variance (PERMANOVA), discriminant analysis, and random forests (RF) classification of the metabolomes of each life stage supervised by historical parental bleaching phenotype enabled the quantification of the vertical transmission of thermal tolerance signatures. Historical bleaching phenotype explained a significant portion of the variation in adults, eggs, sperm, embryos, larvae, and juveniles (PERMANOVA p ≤ 0.001; Fig. 2A). Life stages after fertilization (embryo, larvae, and juvenile) had especially strong signatures of parental bleaching phenotype with RF classification accuracy of 100%. Alpha-diversity (Shannon entropy) was also significantly higher in adults, embryos, larvae and juveniles from parents who bleached compared to those who did not bleach (Fig. S2). Along with the signature of historical bleaching phenotype (i.e., bleaching resistant or bleaching susceptible), there was also a signature of the parental genotype in the gametes (PERMANOVA, Egg p ≤ 0.001; Sperm p ≤ 0.001; Fig. 2A). We mapped metabolites to the symbiont or host fraction by aligning MS/MS spectra to data from purified Symbiodiniaceae pellets or bleached M. capitata corals (see methods). This approach helps partition the source of bleaching phenotype signatures within the holobiont while conservatively assigning a fraction of metabolites to an uncertain ‘both’ category that was not used in this analysis. As defined in the methods below, our metabolite mapping approach identified 571 unique compounds as found in the algae and 579 compounds unique to host tissue. This analysis showed that the transgenerational thermal tolerance signatures were present in both host-associated and symbiont-associated components of the metabolome at every life stage (Fig. 2B,C).

Molecular Families Driving Transgenerational Signatures of Thermal Tolerance

To further explore the biochemistry of bleaching resistance, we first analyzed the metabolome data at the molecular family level after spectral classification with CANOPUS²⁸. At the subclass level, sesquiterpenoids, bile acids, triterpenoids, tetraterpenoids, steroid esters, and amino acids and peptides were more abundant in samples associated with historically bleached corals across all life history stages (Fig. S3). Glycerophospholipids were more abundant in progeny from thermally resistant corals (Fig. S3). These trends were not as evident in sperm samples however, with the most consistent molecular family signatures of bleaching history seen in samples after fertilization. This analysis showed that diverse molecular families reflect historical bleaching phenotype through M. capitata reproduction. Corals that had previously resisted bleaching had a higher abundance of membrane phospholipids and betaine lipids while corals with of a history of bleaching had a higher abundance of steroids and terpenoids.

Lipid Biochemistry Reflecting Thermal Tolerance is Vertically Transferred to Coral Offspring

Variable importance calculations for each metabolite derived from RF classifications were used to identify which metabolites were most strongly associated with the differences in parental phenotype at each life history stage (Fig. S4). The strongest classifier in adults was a lyso-betaine lipid with a fully saturated fatty acid tail (DGCC 16:0, Fig. S4). Because this molecule and other DGCC lipids were in the top 10 classifiers of all life stages after fertilization and third in juveniles, we further explored the biochemistry of this lipid group. Betaine lipids in marine algae have three different head group structures: diacylglyceryl-N-trimethylhomoserine (DGTS), diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) and diacylglycerylcarboxyhydroxymethylcholine (DGCC) forms. The first two are isomers, difficult to distinguish through MS/MS analysis, and the latter is highly abundant in M. capitata but more poorly characterized biochemically^29–32. The most abundant DGCC lipid detected using our LC-MS methods (Lyso-DGCC 16:0) reflected bleaching history in all stages of coral reproduction, except sperm, where it was scarcely detected due to the aposymbiotic nature of this life stage (only detected in 9.5% of sperm samples, at a level 514 times less abundant on average than in egg tissue, Fig. 3A). Characterization of the polarity and fatty acid chain saturation of the DGCC lipid group demonstrated associations with bleaching history phenotype. The hydrophobicity score (retention time x abundance) of DGCC lipids detected with our LC-MS/MS methods, total abundance of those with fully saturated fatty acid tails, and the total abundance of monoacylated forms were all significantly higher in thermally resistant corals (Fig. 3C, E-G). More unsaturated DGCC lipids (DGCC 16:0/22:6, for example) and collective diacylated forms were significantly more abundant in thermally susceptible corals but had fewer clear links to parental phenotype overall (Fig. 3B, D). Finally, in samples of developing larval, the DGCC lipid patterns reflecting thermal tolerance were also present through time, including an increasing abundance of the Lyso-DGCC 16:0 as larvae matured (Fig. 3H). In contrast to the DGCC lipids, DGTS and DGTA did not reflect bleaching history phenotype and were less abundant in the samples overall (Fig. S5).

Phosphocholine (PC) lipids, including mono-alkyl-PCs, such as the host derived platelet activating factor (PAF), were also important variables for bleaching history classification (Fig. S4), and were therefore also explored and compared to the betaine lipid biochemical trends. Similar to the betaine lipid biochemical patterns from the symbiont, thermally resistant corals also had higher levels of PAF 16:0, which was particularly clear in embryo and larvae samples, but not in adult or juvenile corals (Fig. S6). Higher levels of hydrophobic PC lipids and total amount of PC lipids showed more broad links to the bleaching resistant phenotype across the lifespan (Fig. S6). This analysis indicates that lipid biochemical properties reflecting parental thermal tolerance from both the host and symbiont are transferred to the next generation of coral progeny.

Association of the metabolome with Symbiodinaceae

Because symbiont communities are tightly linked to bleaching susceptibility³³, the symbiont communities present in the adult parent corals used for reproduction in this study were identified using the internal transcribed spacer 2 (ITS2) marker gene sequencing. Corals in this study contained Symbiodiniaceae from the genus Cladocopium (n = 22), Durusdinium (n = 16), or a mixed community containing both genera (mixed, n = 9). We compared the metabolomic profiles of coral samples (adult, egg and sperm combined) hosting these communities (C, D, and mixed) and found they were significantly different from each other (PERMANOVA < 0.001; Fig. 4A). This indicates that the algal lineage hosted by the coral has a strong effect on the metabolome profiles across sample types. We also examined relationships between DGCC betaine lipid biochemistry and symbiont genus and that betaine lipid DGCC family showed stark contrasts in its lipid biochemistry between Durusdinium and Cladocopium, particularly the saturation state of these molecules (Fig. S7), demonstrating the strong effect of symbiont on the DGCC biochemistry in parents and gametes.

The association between symbiont and bleaching phenotype in M. capitata in Kāneʻohe Bay is strong but not absolute. During the 2015 bleaching event, a substantial number (31.8%) of colonies hosting thermally susceptible Cladocopium resisted bleaching. The metabolome of corals hosting Cladocopium maintained a strong signature of bleaching susceptibility independent of Durusdinium (PERMANOVA p = 0.006, Fig. 4B). The total sum of DGCC lipids detected with our LC-MS methods, hydrophobicity score, and sum of monacyl forms detected were significantly higher in non-bleached corals and eggs than those that did bleach, while diacyl forms detected with our LC-MS methods were more abundant in eggs and parents that were bleaching sensitive (Fig. 4C). This analysis supports DGCC lipid biochemistry as a signature of coral thermal tolerance independent of symbiont genus. Even in corals hosting symbionts with inherently lower abundances of these molecules, they were still more abundant and highly saturated in those that are thermally tolerant.

Here we show that biochemical modifications associated with a coral’s response to environmental heat stress are passed across generations from parents to offspring. This signature of bleaching phenotype was particularly strong in post-fertilization life stages including embryos, free swimming-larvae, and newly settled juveniles, although it was also present in eggs and sperm to a lesser extent. Importantly, these signatures were identified in partitioned components of both the host cnidarian and dinoflagellate symbiont’s metabolomes. The maintenance of a coral’s biochemical signature of thermal tolerance through all stages of metamorphosis supports the notion that these signatures are a firmly static component of its phenotype and life history. Our data supports the biochemical imprinting of past stress events through coral life history stages and across generations, which has many implications for reef restoration and management efforts in a changing climate, particularly facilitating the selection of thermally resistant stock.

One of the strongest signatures of parental thermal bleaching phenotype was imprinted in the saturation state and abundance of DGCC betaine lipids, a group of unique algal lipids previously associated with bleaching tolerance in adult M. capitata¹⁰. High levels of acyl saturation in the DGCC lipid group, but not the related forms of betaine lipids with different headgroups, make them potentially strong biomarkers of thermal tolerance in this coral species and suggests a functional role in the maintenance of the coral-algal symbiosis under thermal stress. The preference for lyso-lipids with saturated monoacyl tails in thermally tolerant colonies is especially notable, as these lipid types are associated with signaling cascades and represent hydrolyzed forms of diacyl membrane lipids. The location and function of lyso-lipids in M. capitata remain unknown, but mounting evidence from this and other studies are pointing to these molecules, particularly the abundant DGCC 16:0, being especially important to coral symbiont physiology and thermal tolerance^10,34. More notably, this same saturated lyso-lipid phenomenon was found in mono-alkyl-PC lipids, which are associated with the Cnidarian host. Lipids are critical for thermal stability in all organisms and are a major defining factor of bleaching phenotypes in this species^10,35–37. This work highlights DGCC betaine lipids and their lyso-lipid forms as a potentially important mechanism by which thermal stress tolerance is transferred and indicates the need for further research on the role of this under-studied lipid type in cnidarian-dinoflagellate symbioses.

Montipora capitata undergoes a maternal-to-zygotic (MTZ) transcriptional transition in early development (~ 4–14 hours post fertilization), shifting to pathways involving cell division and biosynthesis³⁸. Before this point, transcriptional patterns reflect maternal expression, indicating that the influence of parental phenotype and genotype in our samples is likely the result of biochemical frontloading rather than active metabolism of the gametes. The dependency of gametes on parental metabolism may also explain the influence of coral genotype on the global metabolome of eggs and sperm, which is supported by the significantly lower distances between parent-gamete pairs relative to unrelated progeny. Regardless of the mechanism, our data shows an imprinting of parent biochemical patterns in gametes and offspring of captively bred corals, which bodes well for selection of those with preferable phenotypes, such as thermal tolerance, for restoration efforts on coral reef ecosystems.

The transgenerational biochemical patterns identified here could be due to several potential mechanisms, including parental genetic inheritance, epigenetic inheritance³⁹, partitioning of parental biochemicals into gametes, vertical transmission of microbial symbionts, or any combination. The question of the effect of Symbiodiniaceae transmission mode is of particular interest, as M. capitata vertically transfer their symbionts to progeny, and the lipidome of Durusdinium and Cladocopium algae differ with higher abundance of lyso-DGCC lipids being characteristic of Durusdinium trenchii³⁴. Despite the variation among these algae reported in their isolated form³⁴ and in symbiosis with corals ¹⁰, it is noteworthy that the phenotype of their lipid biochemistry is maintained through all stages of its host’s metamorphosis during reproduction. Furthermore, the same biochemical signature of increasing DGCC lipid content and saturation state in non-bleached colonies is also observed in Cladocopium-dominated corals, which tend to be less thermally tolerant. It is highly likely that vertical transmission of symbionts is a contributing factor to the metabolome/lipidome profiles observed in this study. Regardless, the transfer of these profiles from M. capitata parents with distinct thermal tolerance phenotypes to their offspring bodes well for the success of ongoing restoration efforts leveraging non-bleaching phenotypes of this species in Kāneʻohe Bay, Hawaiʻi and for other vertically transmitting corals worldwide. Furthermore, the potential for direct passage of biochemicals from parents to gametes and offspring provides yet another possible mechanism for acclimatization via transgenerational plasticity in addition to those previously proposed such as DNA methylation and histone modification⁴⁰. Future research will require long-term experiments spanning several generations to definitively elucidate the mechanisms and pathways involved in the production of these transgenerational biochemical signatures linked to prior environmental conditions. If these mechanisms can be identified they could be targeted for assisted evolution and natural selection to better equip corals of the future for the increasing threat of climate change^41,42.

Experimental Framework

All corals in this experiment were part of a model population established during the 2014 and 2015 bleaching events in Hawaiʻi, which created widespread, variable bleaching in Kāneʻohe Bay. Reproductively capable colonies (n = 47) originally assigned bleaching phenotypes in 2014 and 2015^43–45were collected from four reefs in 2020 and returned to the Hawaiʻi Institute of Marine Biology. Colonies were initially tagged in adjacent pairs to minimize environmental effects; 26 colonies demonstrated no bleaching in 2014/2015 (denoted ‘non-bleached’), while the remaining 21 colonies were simultaneously fully bleached (denoted ‘bleached’). All colonies were visibly healthy at the time of this experiment.

Coral Reproduction and Gamete Capture

All samples were collected during a single spawning event on July 8, 2021. Reproductively capable colonies were netted in tanks at the Hawaiʻi Institute of Marine Biology using conical mesh nets and collection tubes that allow for the collection of gametes from a single, individual parent. Montipora capitata is a vertically transmitting hermaphroditic species, so symbiont-provisioned eggs and sperm are contained in the gamete bundles released by each colony. The remaining gametes were recombined into two pools, exclusively bleaching resistant parents, and exclusively bleaching susceptible parents, and then fertilized and reared following established best-practices^25,46,47. Coral reproduction to the juvenile state took approximately 2.5 months and juvenile corals were sampled on October 28, 2021. Adult parents were retrospectively sampled on September 10, 2021.

Sample Collection and Metabolite Extraction

Once egg and sperm bundles began to dissociate, quintuplicate samples of eggs were collected by washing with filtered (0.02 µm) seawater through a 40 µm mesh filter (Fisher Scientific, Waltham MA, USA). Sperm were collected via sequential pelleting and washing in a centrifuge. After separation of sperm and eggs, five 25 µL replicates of each cell type were pipetted into 250 µL of 70% methanol, kept on ice for 30 min, and then stored at − 80°C until shipping. Methanol extractions were shipped on dry ice to Michigan State University for untargeted metabolomics analysis.

Five samples of fifteen embryos each were collected at 15 hours post fertilization (hpf) and developing larvae were collected at 40, 72, 96, and 120 hpf. All samples were collected in quintuplicate from each phenotype pool. At 120 hpf, all remaining larvae were settled on preconditioned aragonite plugs and maintained in flow through ambient seawater systems under natural light. Juveniles from each phenotype pool were carefully removed from plugs with a 12-gauge needle to avoid sampling any of the underlying substrate. Adult corals were sampled with a 5mm dermal curette following the methods outlined in¹⁰. This sampling was conducted after coral spawning to avoid contamination of adult samples with gametes within tissues. All sample types were immediately placed into glass vials containing 250 µL of 70% methanol, kept on ice for 30 min and, then stored at − 80°C until shipping. Methanol extractions were shipped on dry ice to Michigan State University for untargeted metabolomics analysis along with gametic samples.

Algal Genotyping

Three tissue samples were collected from each spawning colony (top, sides) using a dermal curette (3mm diameter) and flash frozen and stored at -80ºC. DNA was extracted using a Quick-DNA Fecal/Soil Microbe kit (ZYMO Research) following manufacturers protocol. Amplicon sequencing to identify Symbiodiniaceae was done by targeting the ITS2 spacer region 2 using the primers ITS-D-For⁴⁸ 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGAATTGCAGAACTCCGTG-3′ and ITS2REV2⁴⁹ 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTCCGCTTACTTATATGCTT-3′ with the Illumina adapters (underlined). Polymerase chain reaction was completed using the KAPA2G Robust PCR Kit (Roche Diagnostics) at the following concentrations and conditions: 1 µL genomic DNA, 11.125 µL DNAse free water, 5 µL Buffer A, 5 µL enhancer, 0.5 µL dNTPS, 1 µL of each primer, 0.25 µL SYBR, and 0.125 µL of Taq polymerase − 30s at 98ºC; 10 s at 98ºC, 30 s at 55ºC, 30 s at 72ºC (x30 cycles); 2 min at 72ºC; 4ºC hold. PCR product was purified using AMPure XP Bead-Based Reagent following manufacturers protocol. Nextera XT indexes and adapters (Illumina) were added using the same recipe as PCR 1 with the following modifications: 2.5 µL PCR product, 6.875 µL DNAse free water, and 5 µL of each Nextera primers, run at 30s at 98ºC; 10 s at 98ºC, 30 s at 55ºC, 30 s at 72ºC (x8 cycles); 2 min at 72ºC; 4ºC hold. Indexed PCR product was cleaned and normalized using Just-A-Plate PCR Purification and Normalization Plates (Charm Biotech). The library was sequenced on an Illumina MiSeq platform (v3 2 x 300bp PE).

Symbiont identity was determined using Symportal⁵⁰ (Symportal.org), which accounts for inter- and intragenomic variance. Data was analyzed in R (4.3.2) using the Phyloseq package. Samples with less than 1500 sequences were culled from the analysis. Parents were assigned as Cladocopium (> 80%), Durusudinium (> 80%), or Mixed. Code and analyses can be found at https://github.com/shaylematsuda/Mcap_parents/tree/master.

Organic extraction and LC-MS/MS data processing

Methanolic extracts were analyzed on a Thermo™ QExactive™ mass spectrometer coupled to a Vanquish Ultra High-Performance Liquid Chromatography (UHPLC) system according to a previously described LC-MS/MS protocol¹⁰. Briefly, chromatography was performed using a mobile phase of 0.1% formic acid in Milli-Q water and acetonitrile with a 12 min-long with linear gradients as follows. Using electrospray ionization, the mass spectrometer cycled through acquisition of a full MS¹ survey scans and then captured tandem MS² mass spectra in positive mode with a scan range set from m/z 100 to 1500. Raw files (.raw) were converted to .mzXML format and all files were processed with MZmine 2.53 software, GNPS feature-based molecular networking, SIRIUS and CANOPUS^27,28,51,52 (details in¹⁰). This provided area under curve abundances for each feature in the metabolome which was then normalized to the total ion signal.

Metabolome Holobiont Mapping

To attribute the metabolites as either algal or host Cnidarian, we used a metabolome mapping approach first employed in¹⁰. This procedure involved the generation of a new metabolome feature table using mzMine 2.53 software with data from this transgenerational coral study and that of previously acquired fully bleached coral and purified algal symbionts. The LC-MS/MS methods were identical for each dataset and the mzMine parameters were modified slightly to ensure proper alignment across the two metabolome batches. Feature-based molecular networking was performed as described above to identify metabolites shared between the two data sets (transgenerational and bleach mapping data). To associate a particular feature as either sourced from the host or the symbiont, the metabolite had to be: 1) shared across the datasets and 2) be on average 10 times more abundant in the bleached coral compared to algal pellet dataset to be considered ‘host’ and vice versa to be considered ‘symbiont’.

Statistical Analysis

All statistics were performed on the total ion chromatogram normalized data in the R or Jump statistical software (SAS Institute Inc.). Alpha-diversity indices were calculated in R and tested between sample types using a Kruskall-Wallis test with post hoc Dunn’s tests between sample types. In addition, the Bray-Curtis distances between egg and sperm samples were calculated from their parent colonies, compared to the distances from non-parent colonies and tested for significance with a Student’s T-test. The entire data set including all life history stages were analyzed for beta-diversity differences between all samples using the Bray-Curtis index and visualized using Principal coordinate analysis plots. Significance between categorical variables among beta-diversity metrics were tested using the permutational multi-variate analysis of variance (PERMANOVA) test reporting both the p-value and variance explained (R²). This included testing among sample types, coral genotypes, and between bleaching history phenotypes. To further explore differences among metabolomes a machine learning classification approach using random forests was also used, reporting the % of samples misclassified by the algorithm. Significant differences in abundance between metabolite features or families between categorical variables was tested with the non-parametric Mann-Whitney U-test.

Acknowledgements

The authors would like to acknowledge funding from the National Science Foundation’s Organismal Response to Climate Change program under grant award number 2307516 to PI Quinn. Coral collections were made under DLNR-DAR permit SAP 2022-22 to the Hawaiʻi Institute of Marine Biology. This is HIMB contribution xx and SOEST contribution xx.

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Transgenerational metabolomic signatures of bleaching resistance in corals

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Abstract

Figures

Introduction

Results

Metabolomic Variation Through the Coral Life Cycle

Transgenerational Thermal Tolerance Signatures in the Coral Metabolome

Molecular Families Driving Transgenerational Signatures of Thermal Tolerance

Lipid Biochemistry Reflecting Thermal Tolerance is Vertically Transferred to Coral Offspring

Association of the metabolome with Symbiodinaceae

Discussion

Methods

Experimental Framework

Coral Reproduction and Gamete Capture

Sample Collection and Metabolite Extraction

Algal Genotyping

Organic extraction and LC-MS/MS data processing

Metabolome Holobiont Mapping

Statistical Analysis

Declarations

Acknowledgements

References

Additional Declarations

Supplementary Files

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