A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea

Abstract

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

Fig. 1.. Progression of abnormal condition affecting D. antillarum.

(A to D) Grossly normal specimens (A) exhibit upright posture and typically shelter beneath rock/coral (photo taken in Pope Point, St. John, April 2022). At condition onset, specimens fall over or stand on spines and exhibit drooping spines (B) (photo taken in Saba, Caribbean Netherlands, April 2022). Advanced stages present as spine loss with elevated predation by fish (C) (photo taken in Aruba, August 2022) and, eventually, death. These signs were also observed in specimens that were experimentally challenged with the Philaster culture FWC2 in aquaria (D). Photo credits: (A) and (D) I. Hewson, (B) A. Hylkema, and (C) D. Behringer.

Fig. 2.. Phylogenetic and microscopic analyses of Philaster sp. in abnormal D. antillarum.

(A to C) Phylogenetic representation of 18S rRNA and 28S rRNA Philaster-like sequences recovered from transcriptomes and 18S rRNA and ITS2 amplified from cultures FWC1 and FWC2 (cultured from abnormal urchins from an affected patch reef off Key Largo, Florida) and USF152 (cultured from an affected urchin during the experimental challenge study).These sequences are compared to homologous sequences of related species obtained from GenBank (accession numbers precede species names). Trees were constructed by maximum likelihood, the Jukes-Cantor substitution model, and with uniform rates of substitution. Bootstrap values of >50 (from 100 replicates) are indicated at nodes. Ciliates with morphologies similar to described Philaster sp. were observed by wet mount light microscopy (Nomarski prism) (D), in the base portion of the spine shaft by histopathology [indicated by arrowheads in (E) hematoxylin and eosin and (F) thionin], and in stained coelomic fluid cytology [indicated by arrowheads in (G); DiffQuick]. Photo credits: (D) to (F) Y. Kiryu and (G) T. Work.

Fig. 3.. Box plots of Philaster-like 28S rRNA loads determined by qPCR for different sample types (body wall, spine base, coelomic fluid, gonad, digestive tract, and >0.2-μm particles collected immediately above urchins) acquired from different specimen types.

The number of specimens surveyed is indicated below box plots. a, b, and c denote groups that are not statistically different (Kruskal-Wallis test with Dunn’s post hoc correction for multiple comparisons). The AAS box plots are shaded gray to highlight results from abnormal specimens. NRS, grossly normal at reference site; NAS, grossly normal at affected site; AAS, abnormal at affected site.

Fig. 4.. D. antillarum response to experimental ciliate challenge.

(A) Proportion of observed grossly normal urchins in control (both <5-μm filtrate and DI water treatments; open circles) and ciliate-challenged treatments (gray circles) following experimental inoculation. (B) Philaster-like 28S rRNA abundance determined by qPCR in discarded spines of ciliate-treated or control urchins at 24, 72, 84, 111, and 168 hours after inoculation. ND, not detected; X, specimen terminated due to spine loss; /, specimens not available because of prior termination; blank indicates no lost spines. Philaster-like 28S rRNA abundance in dissected tissues of terminated specimens (C) and in >0.2-μm aquarium water particulate matter (D) of grossly normal (open box) and abnormal (gray box), ciliate-inoculated urchins. No ciliates were detected in body compartments of any of the control specimens by histopathology.