Scuticociliate Detection and Microbiome Composition in Museum Collections of Diadema Antillarum

BackgroundThe mass mortality of the long-spined sea urchin Diadema antillarum has caused widespread ecological changes across Caribbean reefs, with recent studies identifying the etiological agent as pathogenic ciliate designated as a D. antillarum Scuticociliatosis Philaster-clade (DaScPc). The origin and ecological trajectory of DaScPc remain unresolved, raising critical questions about whether it represents a novel introduction or a resident commensal symbiont that transitioned into pathogenicity. MethodsTo address this, we tested 50 individual preserved museum specimens of D. antillarum collected between 1960 and 2020, with targeted PCR amplification of ciliate 18S, 28S, and 5.8S/ITS rRNA genes for spine, body wall, and coelomic fluid samples (n=100). Following up on recent work that identified bacterial biomarkers of DaSc, we also characterized the microbial communities associated with these museum specimens using 16S rRNA amplicon sequencing. Results. Our results reveal the presence of identical DaScPc 18S rRNA sequences in 21% of tested samples, 28S rRNA PCR yielded sequences at 96-98 % nt identity in only 2% of the tested samples, and we got no amplification from the 5.8S/ITS region. While these findings suggest possible long-term persistence or repeated emergence of this ciliate, the lack of 28S rRNA matches and lack of detection of ITS2 demonstrates that DaScPc 18S rRNA gene detections may be false positives for the ciliate over a highly conserved rRNA region. The microbial composition of the samples didnt yield any of the previously identified disease-associated bacterial biomarkers and showed large shifts in the overall microbial community based on collection period and the facility where the samples are housed. This study demonstrates that museum-preserved echinoderm tissues retain ecologically informative microbial DNA and establishes a molecular framework for disentangling pathogen provenance and its caveats. It also highlights the value and limitations of natural history collections in reconstructing marine disease ecology.