Larvae display conserved gene expression response to heat stress
Larval gene expression (GE) was quantified to assess if plastic responses in gene expression to heat stress varied depending on site of origin or parental identity. Larval survival under heat stress varied between crosses, with larvae produced from dams sourced from far Northern GBR sites exhibiting higher thermal tolerance (Fig. 1b). The cross with the lowest thermal tolerance (LSxSB) did not have any larvae survive the heat treatment (Fig. 1b, Supplementary Fig. 2). GE was examined in aposymbiotic larvae experiencing ambient conditions prior to the heat treatment (“pre”), larvae after exposure to simulated heat stress (35.5 °C for 56 hours, “post heat”), and a simultaneous control temperature of 27 °C (“post ambient”). Therefore, the “pre” larval treatment provided transcriptomic baselines of GE between genetic crosses while “post heat” and “post ambient” comparisons show a baseline for cross-specific heat responses without the confounding effect of symbiosis found in the post-metamorphic phase. Using a principal coordinates analysis (PCoA), we find that GE patterns in larvae were driven by treatment (“pre”, “post ambient”, “post heat”), explaining 29.2% of the variation in survival (padonis < 0.001, Fig. 1a, Supplemental Fig. 2). Discriminant function of principal components (DAPC), a superior method compared to PCoA for partitioning the variance between populations17, recapitulates PCoA patterns showing both treatment and cross-specific effects on larval GE. Using DAPC, we observe that “pre” treatment GE often lies midway between both of the post-treatment GE responses along DAPC-1 (Fig. 1a). Weighted gene co-expression network analysis18 identified groups of genes (the grey60 module, 2069 genes, and the blue module, 1737 genes), which were positively correlated with larval heat treatment (R2 = 0.84, p = 3e-26, and R2 = 0.94, p = 2e-44, respectively.) (Fig. 1c, Supplementary Fig. 3), but did not strongly correlate with larval survival under heat stress, which varied by cross (Fig. 1, Supplemental Fig. 3). This indicates a relatively conserved gene expression response to heat between crosses, despite differences in larval survival. The blue and grey60 modules also showed negative and positive correlations, respectively, with survival under ambient conditions (Supplementary Fig. 3). Gene Ontology (GO) categories enriched in genes within the blue module include “regulation of response to stress” and “nf-kappaB signaling”, while the grey60 module has GO enrichment of “protein folding” and “response to endoplasmic reticulum stress”, categories which are also enriched under a range of environmental stress responses16 and implicated in the selective breeding for heat tolerance4, suggesting their critical importance in a heritable response to heat tolerance acquisition (Supplementary Data 10).
a Discriminant analysis of principal components (DAPC) with heat treatment symbolized by color and cross ID symbolized by shape. The first linear discriminant axis separates the heat treatments, 27 °C for “pre” (n = 33 pools of 10 larvae each) and “post ambient” (n = 33 pools of 10 larvae each), 35.5 °C for “post heat” (n = 30 pools of 10 larvae each), with density plots showing the distribution of samples in each cross and treatment along LD1, ranked from low to high survival in heat stress. b Larval survival in heat and ambient treatments. c Eigengene expression for two WGCNA modules positively, and significantly correlated with larval response to heat. For each cross, the origin of dams is denoted first, and the origin of sires is second (n = 3 pools of 10 larvae each per cross per treatment). Boxplots include the median values (center lines), upper and lower quartiles (box limits), 1.5× interquartile range (whiskers), and outliers (points).
This cross-site breeding design allowed us to further clarify the impact of the local reef, and thus the environmental history, on the molecular responses to thermal stress in coral offspring. Cross identity explained 29.9% of the variation in larval GE (Supplemental Fig. 2, padonis < 0.001), suggesting host genetics plays a vital role in larval heat tolerance. Larvae produced from maternal colonies from the warmest site, Curd Reef, which also exhibited high survival to heat stress (CUxBK, survival 81.25% ± 4.211, Fig. 1b), demonstrated unique larval GE responses under elevated temperature. In the CUxBK cross, we observed an upregulation of thermal response genes (genes in the grey60 module) under “pre” and “post ambient” conditions, indicative of genetic assimilation (Fig. 1c)19, a type of response that has been described in heat-tolerant corals sourced from shallow lagoons in other reef locations20. A cross with the paternal contribution from Curd (LSxCU) exhibited intermediate GE responses to heat that were between crosses with Curd dams and the other crosses produced from cooler reefs (Fig. 1a), further indicating that individual colonies from Curd reef may be differentially adapted to heat stress given their northern, inshore location. Specifically, the expression of 11 genes differentiated larvae with Curd dams in their responses to heat, including an nf-Kappa-B inhibitor (Supplementary Fig. 4). Additionally, modeling in DESeq2 of the “pre heat” larval samples with survival under subsequent heat stress identified 438 genes predictive of larval survival in A. tenuis (Supplementary Data 8), which are further referenced throughout as “thermal tolerance” genes. GO categories “cell cycle processes” and “ribosome” were significantly enriched in genes that were predictive of larval survival (Supplementary Data 5). The association of these genes in crosses produced from parents of Curd origin suggests that these genes may play a role in the local adaptation to the more extreme, inshore, warmer conditions experienced by this reef.
Juvenile gene expression varies based on symbiont community
While mechanisms elucidating acquired heat tolerance have been examined in aposymbiotic larvae3,4 (Fig. 1, Supplementary Figs. 2–4), no studies have yet resolved GE responses to heat or variable symbiotic states in juvenile corals. Arguably, quantifying the role of population origin, plasticity, and symbiosis in this critical life-history stage is urgently needed as juvenile survival and sensitivity to environmental stress are key for the conservation and restoration of coral populations globally. To address this, larvae from the aforementioned crosses were induced to metamorphose and then subsequently exposed to a range of cultured or wild Symbiodiniaceae treatments. Juveniles were infected with cultured Cladocopium goreaui (“C1”), Durusdinium trenchii (“D1a”), a heat-evolved species of Cladocopium goreaui (“SS1”)21, or a diverse community of putatively heat-tolerant symbionts from the sediments collected from Curd (“SED”), with the dominant infection of each symbiont type confirmed with transcripts mapping to each genus of Symbiodiniaceae (Supplementary Fig. 6). Juvenile Acropora uptake diverse communities of Symbiodiniaceae from their environment, a dynamic process comprised of a taxonomically variable symbiont consortium that plays a substantial role in heat tolerance acquisition and survival in juveniles10,22. At ambient temperatures, juvenile host GE is mainly clustered by symbiont identity although a significant effect of the cross was also observed (Fig. 2). A permutational multivariate ANOVA revealed that the cross explained 15.7% of the variance in juvenile GE under ambient conditions with symbiont treatment explaining 10.6%. In partitioning the variance using DAPC, we find that two crosses (DRxDR and BKxBK) showed unique GE clustering (Fig. 2) and despite close proximity of these two sites on the offshore GBR, appear to have unique histories of selection and acclimation. Juveniles in the “SED” treatment hosted a highly diverse symbiont community, comprised of >300 taxonomic groups11, which may explain the pattern of high GE divergence in these juveniles compared to those hosting cultured symbionts. This emphasizes the influence of symbiont diversity in the modulation of host physiology, even during early ontogeny (Fig. 3, Supplementary Fig. 6). Moreover, juveniles in the ambient “SED” treatment exhibited the downregulation of genes associated with signal transduction, coenzyme transport, and secondary metabolite synthesis, and upregulation of energy production and conversion, and intracellular trafficking, secretion, and vesicular transport (Fig. 4a, Supplementary Data 9), compared to juveniles with cultured symbionts. These functional categories are a hallmark of host regulation of the symbiotic state23, likely in the midst of the winnowing process24,25, and are most likely representative of juvenile host gene expression in situ on the reef. In juveniles, gene modulation in response to different symbiotic states included changes in genes associated with cell receptor signaling, cellular adhesion proteins, transcription, cellular stress responses, immunity, and the cell cycle (Fig. 4a, Supplementary Data 6). Juveniles hosting the SS1 strain, which was experimentally evolved to tolerate higher temperatures, upregulated genes associated with biological adhesion molecules, and regulation of cell morphogenesis (Supplementary Data 6). These functional categories of genes have been previously implicated as signatures of selection and convergence in the evolution of vertical transmission in corals26, suggesting that these pathways are specifically modulated by the host when the symbioses are perturbed.
Discriminant analysis of principal components (DAPC) on gene expression of juveniles hosting different symbioses under ambient conditions. DAPC was performed on variance stabilized data (VSD) grouped by cross+symbiosis (a) and with the effect of cross removed by limma (b). For each cross, origin of dams is denoted first, and the origin of sires is second.
Discriminant analysis of principal components (DAPC) on gene expression of juveniles hosting different symbioses under ambient and heat conditions. DAPC was performed on variance stabilized data (VSD) grouped by cross+treatment+symbiosis (a) and with the effect of the cross removed by limma (b) with density plots showing the distribution of samples in each symbiosis and treatment along LD1.
Hierarchical clustering analysis of KOG enrichments in juveniles hosting different symbioses at ambient conditions (a) and host responses to heat in juveniles hosting different symbioses (c). Correlations of KOG delta rank values for each comparison under ambient conditions (b) and symbiosis responses to heat (d). KOG categories in bolded squares denote the significance of Mann–Whitney U test at padj < 0.05. Exact Benjamini-Hochberg adjusted p values for each comparison are in Supplementary Data 9. Color scale in a and c are KOG delta rank values.
Larvae and juveniles have different responses to heat stress
Under heat stress, juveniles hosting C1 experienced substantial mortality and are thus not represented in this study, while juveniles hosting SS1, experimentally evolved from C1, exhibited higher survival under heat stress. Survival of juveniles hosting SS1, D1a, and communities from the sediments all exhibited similarly high survival under heat stress11 as well as significantly positively correlated functional responses to heat (Fig. 4d). While juveniles hosting SS1 at ambient conditions hosted a majority of Cladocopium transcripts, SS1 juveniles under heat stress all exhibited elevated proportions of Durusdinium-derived transcripts (Supplementary Fig 6). Therefore, the heat stress itself may have promoted a shift towards Durusdinium-derived transcripts in SS1 infected juveniles, potentially facilitating their thermal tolerance. Further, juveniles hosting SS1 showed positively significant molecular responses to heat that are indicative of corals hosting D1a as seen in the KOG correlations between these two responses (R2 = 0.74 p = 8.5e-05, Fig. 4d), with stronger correlations to those hosting communities from the sediments (R2 = 0.8 p = 9.2e-06, Fig. 4d). Despite these overall functional similarities, key differences were observed that highlight the unique interactions between symbiont identity, symbiont community, and coral host origin that contribute to juvenile heat stress responses. For example, under heat stress, SS1 juveniles significantly downregulated cell cycle control, cell division, and chromosome partitioning genes compared to the other two symbioses (Fig. 4d), indicative of a trade-off between growth and thermal tolerance. Compared to the other symbioses, D1a juveniles downregulated signal transduction genes (Fig. 4d), genes likely to be involved in symbiosis establishment and maintenance.
Heat-stress responses in juveniles hosting all symbiont treatments exhibited non-significant correlations with the heat stress responses in larvae (Fig. 4d). This underscores that heat tolerance is likely regulated by different molecular mechanisms in larvae and juvenile Acropora tenuis. Juveniles in all symbiont treatments showed strong upregulation of energy production and conversion genes compared to the larval heat stress response (Fig. 4c). Larval heat tolerance also showed no functional correlations with either larval or juvenile responses to stress, suggesting different molecular mechanisms confer tolerance and response in early life-history stages.
Source: Ecology - nature.com
