Results
Transmission experiment
There was a significant difference in the number of days to the onset of
visible disease lesions between species (ANOVA:F 1,29 = 6.22, p < 0.019), withO. faveolata significantly more likely to develop SCTLD lesions
compared to M. cavernosa (log-rank: z 1,29= 3.569, p < 0.001; Figure S1). Transmission rates in
the disease-exposed treatment at the conclusion of the experiment were
95% for O. faveolata with lesions occurring at a mean of 3.1 ±
0.5 days, while 55% of M. cavernosa fragments exhibited lesions
at a mean of 5.4 ± 1.0 days (Table 1). No controls in either species
developed lesions. Signs of SCTLD were similar between species, with
tissue necrosis and lesion formation/progression being the most common
(Dataset S1).
Two-way PERMANOVAs identified significant effects of coral genotype and
disease status for M. cavernosa and associated Cladocopiumdatasets, while the effects of genotype and exposure group were
marginally insignificant for O. faveolata (p = 0.057 andp = 0.056, respectively) and nonsignificant for associatedDurusdinium symbionts (Table 3). Pairwise differential expression
tests with DESeq2 indicated the majority of differentially
expressed genes (DEGs) to be attributed to diseased versus healthy
samples for M. cavernosa (3,890 upregulated and 3,878
downregulated), with 1,820 DEGs between diseased and samples not
exhibiting visible disease signs following exposure (NAI; 960
upregulated and 860 downregulated), and 115 DEGs between NAI and healthy
samples (43 upregulated, 72 downregulated). Few DEGs were identified for
any pairwise comparisons in Cladocopium symbionts, with five in
diseased versus healthy samples and two in NAI versus healthy samples.Orbicella faveolata showed moderate numbers of DEGs between
diseased and healthy samples (385 upregulated and 188 downregulated),
with no DEGs for Durusdinium (Table 4). Ordination with PCoAs
demonstrated that diseased samples were distinct from all other samples
for M. cavernosa , while NAI and healthy samples were more similar
(Figure 1). This relationship was reversed for Cladocopium data,
where diseased and NAI samples were more closely related to each other
than to healthy samples. Both O. faveolata and Durusdiniumsamples showed similarities between healthy and diseased samples, with
little differentiation between exposure groups in the ordination space
(Figure 1).
Functional enrichment analysis using gene ontology (GO) identified 434
GO terms across three categories (molecular function [MF]: 38;
biological process [BP]: 287; cellular component [CC]: 109) for
diseased versus healthy M. cavernosa samples, and 45 GO terms forO. faveolata (MF: 9; BP: 20; CC: 16; Table S1). The majority of
enriched GO terms were negatively represented in diseased M.
cavernosa , while the opposite was true for O. faveolata . For
diseased versus NAI M. cavernosa , 371 GO terms were significantly
enriched (MF: 50; BP: 244; CC: 77), while only 8 terms were
significantly enriched for NAI versus healthy samples (BP: 2; CC: 6;
Table S2). No GO terms were significantly enriched forCladocopium or Durusdinium symbionts, however. Functional
enrichment of eukaryotic orthologous groups (KOGs) demonstrated similar
patterns between diseased versus healthy and diseased versus NAI samples
for M. cavernosa (Figure S2), where metabolic and transport
mechanisms were generally downregulated in diseased samples, and nuclear
structure and translation-related pathways were upregulated. Some
nuclear structure and membrane pathways were positively enriched in NAI
versus healthy M. cavernosa , with down regulation of translation,
cytoskeleton, and extracellular pathways. Cladocopium diseased
and NAI samples showed mild enrichment of most pathways relative to
healthy controls, with nuclear structure as the most positively
enriched, and cell motility as the most downregulated. Similar trends
were observed for diseased versus healthy O. faveolata as
compared to M. cavernosa , while Durusdinium showed an
inverse pattern of KOG enrichment in diseased samples relative toCladocopium (Figure S2).
Weighted gene coexpression network analysis (WGCNA) identified ten
modules with significant correlations to experimental factors (disease
status and the time to onset of lesions). In general, modules that were
negatively correlated with healthy samples were positively correlated
with diseased samples (Figure S3). Several modules were also correlated
with NAI samples that demonstrated similar expression to diseased
samples (‘darkorange2’) and the time to transmission (‘thistle1’ and
‘darkorange2’), or inverse relationships with healthy samples
(‘navajowhite1’). Of the modules correlated with NAI samples, only
‘darkorange2’ showed significant GO enrichment to the mRNA metabolic
process (BP) and peptidase, cytosolic, and ribosomal complexes (CC;
Table S4). Four modules were found to have significant trait
correlations for Cladocopium , with similar expression patterns
between NAI and diseased samples relative to healthy controls in the
‘pink’ module, but no significant GO enrichment (Table S4). The O.
faveolata dataset had six modules with significant correlations to
disease status and the time to onset of lesions; most of these were
inversely related between diseased and healthy samples, apart from the
‘tan’ module that was inversely related between NAI and healthy samples
(Figure S3). There were no significantly enriched GO terms for the
‘pink’ or ‘tan’ modules correlated with NAI samples. WGCNA was not
successful in assigning modules to the Durusdinium dataset.
Module hub genes (i.e., genes with the highest module membership) were
largely unannotated for the respective reference datasets, with no
apparent relationship between coral species or among associated
symbionts (Table S3).
Orthofinder identified 681 orthologous DEGs in diseased versus healthy
samples; of these, 438 showed similar expression patterns between
species (272 upregulated and 166 downregulated), and 243 showed inverse
expression (Figure 2). While expression of individual genes was in some
cases inversely related between species, the corresponding KOG
annotations demonstrated that the number of DEGs within each KOG class
that was up- or downregulated was relatively similar, suggestive of an
overall similar function response to disease exposure between M.
cavernosa and O. faveolata . Likewise, examination of GO
categories in diseased versus healthy samples indicated 21 GO terms (MF:
2; BP: 11; CC: 8) that were commonly enriched in the same direction
between species (Table 5; Figure 3).
Intervention experiment
PERMANOVAs indicated no significant effect of time or treatment on the
gene expression of M. cavernosa or associated Cladocopiumsymbionts (Table 3). Visualization with PCoA revealed overlapping sample
dispersion in the coral and symbiont ordinations (Figure 4).
Differential expression analyses, however, identified DEGs associated
with pairwise treatment/time comparisons, with a high number of DEGs
between diseased and healthy corals at t0 (410 DEGs: 284
upregulated and 126 downregulated), as well as between
amoxicillin-treated corals at t1 and their original
diseased state at t0 (241 DEGs: 48 upregulated and 193
downregulated; Table 4; Figure 4). There were few differences between
treated and healthy corals at t1 (5 DEGs: 4 upregulated
and 1 downregulated) and temporal shifts in gene expression were evident
(e.g., 248 DEGs in t1 versus t0 healthy
corals). Few DEGs were identified across pairwise comparisons forCladocopium (Table 4; Figure 4).
Functional enrichment analysis identified 46 significantly enriched GO
terms in diseased versus healthy corals at t0 (MF: 9;
BP: 10; CC: 27), 39 GO terms in treated versus diseased corals (MF: 6;
BP: 14; CC: 19), and 7 GO terms in treated versus healthy corals at
t1 (MF: 1; CC: 6; Table S5). Similar to M.
cavernosa samples from the transmission experiment, the majority of
enriched GO terms were negatively enriched in diseased versus healthy
corals. Conversely, treated corals showed positive enrichment of most GO
terms relative to healthy controls at t1. KOG enrichment
in diseased versus healthy corals at t0 was similar to
the trends observed for the transmission experiment, where several
metabolic and transport pathways were negatively enriched (Figure S4).
Notably, the nuclear structure KOG class was highly negatively enriched
in diseased corals, where it was shown to be positively enriched for
both diseased M. cavernosa and O. faveolata samples from
the transmission experiment. Comparison of treated versus diseased
corals demonstrated an opposite response in the same pathways as
compared to diseased versus healthy corals, showing an increase in
metabolic, transport, and nuclear structure classes. No KOG classes were
strongly enriched when comparing treated and healthy corals at
t1. Cladocopium KOG enrichment was similar in
diseased versus healthy corals to the transmission experiment samples,
with strong positive enrichment of the nuclear structure class and
negatively enrichment of cell motility. Following treatment with
amoxicillin, those patterns were reversed, even when comparing treated
to healthy corals at t1 (Figure S4).
WGCNA returned 8 modules significantly correlated with treatments/time
points, with the strongest patterns of correlation reversal between
healthy corals at t0 to t1 (modules
‘darkred’ and ‘salmon;’ Figure S5). Modules ‘greenyellow’ and ‘purple’
showed inverse correlations between diseased and healthy corals at
t0, and only the ‘pink’ module showed an inverse
relationship between treated and diseased corals. Only the ‘darkred’
module contained significantly enriched GO terms, all of which were
related to endoplasmic reticulum complexes (CC; Table S4). One module
had a significant correlation to experimental traits inCladocopium , but it was driven by one sample and therefore
disregarded. Visualization of expression patterns for the 30 most
significant DEGs in modules ‘greenyellow’ and ‘grey60’ demonstrated a
shift in expression patterns from the original disease state being
distinct to healthy controls, to overall similarities between treated
and healthy corals (Figure S5). Of the module hub genes with
annotations, none matched among reference transcriptomes for the
transmission experiment, although the ‘green’ module hub gene belongs to
the heat shock protein 70 family (Table S3).
Cross-experiment comparisons
Comparison of diseased versus healthy samples from the transmission and
intervention experiments revealed 1,458 genes that were shared between
experiments. The majority of common genes showed the same directionality
between experiments (452 upregulated and 489 downregulated), while 517
genes showed inverse relationships between experiments (Figure 5). When
comparing diseased versus healthy samples in the transmission experiment
to treated versus diseased corals in the intervention experiment,
however, the majority of genes were found in inverse relationships
between experiments (1,041 genes), whereas only 285 genes were found to
share the same direction of expression (121 upregulated and 164
downregulated). Gene representation within KOG classes was largely
similar across experiments, with the exception of unannotated genes that
contributed to variability between experiments and among disease
status/treatment comparisons (Figure 5).