RNA-Seq of the Caribbean reef-building coral Orbicella faveolata (Scleractinia-Merulinidae) under bleaching and disease stress expands models of coral innate immunity

Abstract

Climate change-driven coral disease outbreaks have led to widespread declines in coral populations. Early work on coral genomics established that corals have a complex innate immune system, and whole-transcriptome gene expression studies have revealed mechanisms by which the coral immune system responds to stress and disease. The present investigation expands bioinformatic data available to study coral molecular physiology through the assembly and annotation of a reference transcriptome of the Caribbean reef-building coral, Orbicella faveolata. Samples were collected during a warm water thermal anomaly, coral bleaching event and Caribbean yellow band disease outbreak in 2010 in Puerto Rico. Multiplex sequencing of RNA on the Illumina GAIIx platform and de novo transcriptome assembly by Trinity produced 70,745,177 raw short-sequence reads and 32,463 O. faveolata transcripts, respectively. The reference transcriptome was annotated with gene ontologies, mapped to KEGG pathways, and a predicted proteome of 20,488 sequences was generated. Protein families and signaling pathways that are essential in the regulation of innate immunity across Phyla were investigated in-depth. Results were used to develop models of evolutionarily conserved Wnt, Notch, Rig-like receptor, Nod-like receptor, and Dicer signaling. O. faveolata is a coral species that has been studied widely under climate-driven stress and disease, and the present investigation provides new data on the genes that putatively regulate its immune system.

Keywords: Cnidaria; Coral; Disease; Innate immunity; RNA-seq.

Figure 1. Representative images of colonies sampled in the present study.

(A) Asymptomatic. (B) Caribbean yellow band-diseased. (C) Partially bleached colonies. (D) Completely bleached colonies. (E) Caribbean yellow band-diseased and bleached. Photos by E. Weil.

Figure 2. Frequency distribution of taxonomic identities of best hits to O. faveolata transcripts.

BLAST2GO analysis results showing taxonomic identities of best BLASTx hits to O. faveolata transcripts from SwissProt non-redundant protein sequence database.

Figure 3. Frequency distribution of selected Gene Ontology (GO) terms annotated to O. faveolata transcripts.

Results of BLAST2GO annotation of O. faveolata transcripts with GO terms. (A) Biological process GO terms. (B) Molecular function GO terms. (C) Cellular component GO terms.

Figure 4. Numbers of unique and shared sequences between the present and (Pinzón et al., 2015) O. faveolata transcriptomes for select immunity-related KEGG pathways.

Results from KEGG KAAS parallel annotation of the present transcriptome and the (Pinzón et al., 2015) transcriptome for O. faveolata. The number of non-redundant annotations mapped to select KEGG pathways for each transcriptome are shown.

Figure 5. Phylogenetic analysis of Wnt-like protein sequences in O. faveolata transcriptome.

Predicted protein sequences from N. vectensis (Nvec), O. faveolata (Ofav), A. pallida (Apall), A. digitifera (Adig), and A. millepora (Supplemental Information 11) were used for maximum likelihood phylogeny estimation with 100 bootstraps.

Figure 6. Modified O. faveolata Notch signaling pathway from KEGG.

Delta-like ligand (DLL), protein jagged (JAG), disintegrin and metalloproteinase domain-containing protein 17 (ADA), O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (FNG), neurogenic locus Notch homolog protein (NOTC), numb-like protein (NMBL), segment polarity protein dishevelled homolog (DVL), E3 ubiquitin-protein ligase DTX1 (DTX), presenilin enhancer protein 2 (PEN), nicastrin (NICA), presenilin-1 (PSN), anterior pharynx defective 1 (APH), E1A/CREB-binding protein (EP300), recombining binding protein suppressor of hairless (RBPJL), SNW domain-containing protein 1 (SNW), C-terminal binding protein (CTBP), hairless (HAIRLESS), nuclear receptor co-repressor 2 (NCOR), histone deacetylase 1 or 2 (HDAC), CBF1 interacting corepressor (CIR), groucho (GROUCHO), hairy and enhancer of split 1 (HES), pre T-cell antigen receptor alpha (PTCRA).

Figure 7. Modified O. faveolata NLR signaling pathway from KEGG.

NLR family CARD domain-containing protein 4 (NLRC), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), suppressor of G2 allele of SKP1 (SUGT1), receptor-interacting serine/threonine-protein kinase 2 (RIPK2), mitogen-activated protein kinase kinase kinase 7 (TAK), TAK1-binding protein 1 (TAB), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase 1 or 3 (ERK), p38 MAP kinase (p38), inhibitor of nuclear factor kappa-B kinase subunit gamma (IKKγ), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), nuclear factor kappa beta (Nfκβ), TNF receptor-associated factor 6 (TRAF), Caspase (CASP), baculoviral IAP repeat-containing protein (BIRC), tumor necrosis factor alpha-induced protein 3 (A20).

Figure 8. Domain architecture of dicer-like protein sequences derived from the O. faveolata transcriptome.

(A) Hmmscan analysis of protein domain architecture for O. faveolata predicted protein sequences. Dicer-like protein with similar domain architectures are also shown (Supplemental Information 12) (B) miRNA and siRNA pathway components present in the O. faveolata transcriptome.

Figure 9. Modified O. faveolata RLR pathway from KEGG.

Ubiquitin thioesterase CYLD (CYLD), ATP-dependent RNA helicase DDX58 (RIG-I), interferon-induced helicase C domain-containing protein 1 (MDA5), dihydroxyacetone kinase (DAK), ATP-dependent RNA helicase DHX58 (DHX58), autophagy-related protein 5 (ATG5), autophagy-related protein (ATG12), Nod-like receptor (NLR), mitochondrial antiviral-signaling protein (MAVS), transmembrane protein 173 (STING), TNF receptor-associated factor 3 (TRAF), mitogen-activated protein kinase kinase kinase 7 (TAK), TAK1-binding protein 1 (TAB), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase 1 or 3 (ERK), p38 MAP kinase (p38), nuclear factor kappa beta (NFKB), mitogen-activated protein kinase kinase kinase 1 (MAPK).