A comprehensive untargeted lipidomic assessment was performed under standard culture conditions (22 ˚C; see methods section for details) to establish a baseline for the three Symbiodiniaceae species: S. microadriaticum (ITS2 type A1), B. minutum (B1) and C. goreaui (C1). A total of 276 compounds were identified and sorted into seven classes (i.e. pigments, membrane lipids as glycolipids, aminolipids, phospholipids and shingolipids; storage lipids and others) (Table 1; Fig. S1), to differentiate their presence among distinct cell compartments. The most significant difference in abundance of lipid classes among the three species was observed in the concentration of membrane lipids (ML) (1 way - ANOVA Fssp. = 51.22; p < 0.01; Table S2) and their esterified fatty acids, which was up to 40% lower in B. minutum than in the other two species (Fig. 1a and b). Most of the lipid compounds (75 out of 86) that significantly differed among the three species were ML components (Fig. S2). Of these 75 compounds, 43 of them were esterified to omega-3 fatty acids, either octadecatrienoic acid (18:4n-3), octadecapentaenoic acid (18:5n-3) or docosahexaenoic acid (DHA; 22:6n-3). Glycolipids were predominantly linked to 18:4 and 18:5, whereas phosphatidylcholine (PC) and 1,2-diacylglyceryl-3-(O-carboxyhydroxymethylcholine (DGCC) were mostly linked to DHA (Table S1). The amounts of membrane bond oxidized PUFA (oxy-PUFA) did not differ significantly among the three species at baseline (Fig.1c).
To assess the influence of heat shock on population density, cultures were sampled over time for both heat shock and control groups of each symbiont. Biomass sampling for lipidomics was performed immediately after the four-hour long heat shock (T4), then 24 hours later (T28) and finally at the end of the experiment at 240 hours (T244). Even though the control of S. microadriaticum had low population density number at T244, no significant differences among the growing control cultures of the three species and the heat shock samples of C. goreaui were noticed (Table S3). On the other hand, heat shock samples of both S. microadriaticum and B. minutum showed an initial reduction to about half of their population, compared to control at T4, thus their growth curves were significantly affected (ANCOVA FHS [Spp] = 26.29; p < 0.01; Table S3) (Fig. 2). The only symbiont that fully ceased growth was B. minutum, indicated by the constant decline in its population density after heat shock (Fig. 2).
Overall alterations in lipidome profiles after heat shock
Thermal susceptibility was defined by changes in the population densities of each symbiont and by the intensity of ML remodeling, where the largest differences, those with ANOSIM R (p < 0.01) values approaching 1, can be visualized using the multidimensional scaling plot (MDS) (Fig. 3a). Breviolum minutum drastically changed its membrane lipidome (R = 0.75) with no recovery at 24 (T28) nor even 240 (T244) hours after heat shock (Fig. 3a), which suggest irreversibly damaged to its cells and/or cell death at T244. Symbiodinium microadriaticum was an intermediate heat tolerant (R = 0.73) and C. goreaui was the most heat tolerant symbiont (R = 0.68). Differences among treatments of both S. microadriaticum and C. goreaui were within 30 Bray-Curtis similarity indexes, independent of heat shock treatment at all monitored times. The same similarity was shared only by the control of B. minutum (c.f. Fig. 3a). Glycolipids, plastoquinone and pigments, together with cardiolipin, PC and the total sum of oxy-PUFA esterified to glycolipids and DGCC were the ML classes that exhibited most variation among Symbiodiniaceae after heat shock according to principal component analysis (PCA; Fig. 3b). The greater distancing of heat shock treated samples of B. minutum indicated in both PCA and MDS (Fig. 3) also suggests that this heat sensitive symbiont suffered the greatest lipidome alterations. DGCC, cholesterol and other membrane components were less robust in explaining heat shock induced lipidome alterations in the Symbiodiniaceae species (cf. Fig. 3b, detailed on Table S1).
Alterations in the heat sensitive symbiont B. minutum
The concentration of glycolipids in heat shock samples of B. minutum was 77% lower than the control sample at T4 (3-way ANOVA FHS. [Spp.] = 11.76; p < 0.01; Table S4 and 2-way ANOVA FHS = 13.46; p < 0.01; Table S5) and such variation may be explained by the reduction of PUFA linked to glycolipids (FHS. [Spp.] = 21.44; p < 0.01; Table S4 and FHS = 26.90; p < 0.01; Table S5) (Fig. 4). At T244, concentration of PUFA linked to glycolipids was 91% lower than control, whereas the concentration of oxy-PUFA linked to glycolipids increased by 20 times compare to the control sample (FTi. [Spp.] = 8.88; p < 0.01; Table S4 and FTi = 17.57; p < 0.01; Table S5). MGDG (18:4/18:5-OH) and DGDG (18:4/18:5-OH) were among the top 20 most relevant altered lipids comparing heat shock and control samples at T244 (Table S6). Their concentrations increased by 1.47 and 1.12 pg, respectively, whereas concentrations of their precursors (MGDG (18:4/18:5) and DGDG (18:4/18:5) dropped to above half at T4 and did not recover at T244 (Table S6). Lysolipids (FHS. [Spp.] = 9.09; p < 0.01; Table S4) and free oxy-PUFA (oxylipins) (FHS. [Spp.] = 4.89; p < 0.01; Table S4) also increased at T244 (Fig. 5c and d). Oxylipins derived from the oxidation of linoleic acid (LA; 18:2n-6), and docosahexaenoic acid (DHA; 22:6n-3), represented by hydroxyoctadienoic (HODE) and hydroxydocosahexaenoic (HDoHE) acids, respectively, were further investigated in oxylipidomics. The most abundant LA-derived oxylipins, independent of heat shock exposure, were 9- and 13-HODE isomers (Fig. S5). Relative percentages of 14- and 19-HDoHE isomers were higher than control only at T244 (Fig. S6). Concentrations of both plastoquinone (FHS. [Spp.] = 15.40; p < 0.01; Table S4) and chlorophyll-a (FHS. [Spp.] = 13.97; p < 0.01; Table S4) were reduced after heat shock (Fig. 5a and b), similarly to glycolipids. The concentrations of PC (FHS. [Spp.] = 34.38; p < 0.01; Table S4), DGCC (FHS. [Spp.] = 33.61; p < 0.01; Table S4) and cholesterol (FHS. [Spp.] = 15.47; p < 0.01; Table S4) decreased after heat shock, whereas sphingolipids (i.e., ceramides, phytoceramides and Hexosyl-ceramides) increased, exhibiting a more than 5-fold gain at T244 (FHS. [Spp.] = 7.49; p < 0.01; Table S4 (Fig. S3). Overall, the storage lipids did not change significantly after heat shock, but the MUFA (FHS = 8.06; p < 0.01; Table S5) content increased in triacylglycerols (TAG) at T244.
Alterations in the heat tolerant symbionts S. microadriaticum and C. goreaui
Although S. microadriaticum and C. goreaui persisted growing after heat shock, their lipidome alterations were drastically different, except for the relative percentages of HODE and HDoHE isomers. As shown in B. minutum, LA-derived 9- and 13-HODEs were also the most abundant isomers in this two symbionts (Fig. S5), whereas HDoHEs proportions were similar after heat shock compared to controls (Fig. S6).
In S. microadriaticum, the concentrations of glycolipids (Fig.4), chlorophyll-a, oxylipins (Fig.5a and d), sphingolipids (Fig. S3d) and storage lipids (Fig. S4) did not change significantly. Plastoquinone, lysolipids (Fig. 5b and c), PC, DGCC and cholesterol (Fig. S3) slightly increased at either T4, T244 or both. In C. goreaui, plastoquinone (Fig. 5b), PC and DGCC (Fig. S3) concentrations decreased similarly, whereas chlorophyll-a decreased at T4, but increased at T244, compared to control (Fig. 5a). Significant decrease in glycolipids concentration at T244 (FHS = 26.90; p < 0.01; Table S5) was only validated by a 2-way ANOVA species-specific analysis representing a drop of 35% compared to the control samples. The concentration of oxy-PUFA esterified to glycolipids was uniquely lower than control at T4 (FTi = 27.61; p < 0.01; Table S5) (Fig. 4). Cladocopium goreaui also had the lowest oxylipins content (FSpp. = 8.16; p < 0.01; Table S4) among all symbionts, independently of heat shock (Fig.4). Lysolipids (Fig. 5c), cholesterol and sphingolipids (Fig.S3) concentrations remained unchanged. Storage lipids concentration increased in C. goreaui at T244 and the greatest change was observed for PUFA acyl chains esterified to TAG (FHS. [Spp.] = 6.62; p < 0.01; Table S5), which were three times greater than the control samples (Fig. S4b).