Interspecific transmission and recovery of TCBS-induced disease between Acanthaster planci and Linckia guildingi.

The susceptibility of the coral-feeding crown-of-thorns starfish Acanthaster planci to disease may provide an avenue with which to effectively control population outbreaks that have caused severe and widespread coral loss in the Indo-Pacific. Injecting thiosulfate-citrate-bile-sucrose (TCBS) agar into A. planci tissues induced a disease characterized by dermal lesions, loss of skin turgor, collapsed spines, and accumulation of mucus on spine tips. Moreover, the symptoms (and presumably the agent) of this disease would spread rapidly intraspecifically, but interspecific transmission (to other species of echinoderms) is yet to be examined. Vibrio rotiferianus, which was previously reported as a pathogen isolated from lesions of experimentally infected A. planci, was also recovered from Linckia guildingi lesions after several days of direct contact with diseased A. planci, demonstrating disease transmission. However, all L. guildingi fully recovered after 31 ± 16 d. Further studies are in progress to understand the ecology of Vibrio infection in A. planci and the potential transmission risk to corals, fishes, and other echinoderms to evaluate whether injections of TCBS could be a viable tool for controlling A. planci outbreaks.

Refined identification of Vibrio bacterial flora from Acanthasther planci based on biochemical profiling and analysis of housekeeping genes.

We used a polyphasic approach for precise identification of bacterial flora (Vibrionaceae) isolated from crown-of-thorns starfish (COTS) from Lizard Island (Great Barrier Reef, Australia) and Guam (U.S.A., Western Pacific Ocean). Previous 16S rRNA gene phylogenetic analysis was useful to allocate and identify isolates within the Photobacterium, Splendidus and Harveyi clades but failed in the identification of Vibrio harveyi-like isolates. Species of the V harveyi group have almost indistinguishable phenotypes and genotypes, and thus, identification by standard biochemical tests and 16S rRNA gene analysis is commonly inaccurate. Biochemical profiling and sequence analysis of additional topA and mreB housekeeping genes were carried out for definitive identification of 19 bacterial isolates recovered from sick and wild COTS. For 8 isolates, biochemical profiles and topA and mreB gene sequence alignments with the closest relatives (GenBank) confirmed previous 16S rRNA-based identification: V. fortis and Photobacterium eurosenbergii species (from wild COTS), and V natriegens (from diseased COTS). Further phylogenetic analysis based on topA and mreB concatenated sequences served to identify the remaining 11 V harveyi-like isolates: V. owensii and V. rotiferianus (from wild COTS), and V. owensii, V. rotiferianus, and V. harveyi (from diseased COTS). This study further confirms the reliability of topA-mreB gene sequence analysis for identification of these close species, and it reveals a wider distribution range of the potentially pathogenic V. harveyi group.

Injection of Acanthaster planci with thiosulfate-citrate-bile-sucrose agar (TCBS). I. Disease induction.

This is the first report of the successful induction of a transmissible disease in the coral-eating crown-of-thorns starfish Acanthaster planci (COTS). Injection of thiosulfate-citrate-bile-sucrose agar (TCBS) culture medium into COTS induced a disease characterized by discoloured and necrotic skin, ulcerations, loss of body turgor, accumulation of colourless mucus on many spines especially at their tip, and loss of spines. Blisters on the dorsal integument broke through the skin surface and resulted in large, open sores that exposed the internal organs. Oedema and reddened digestive tissues and destruction of connective fibers were common. Moreover, healthy COTS in contact with these infected animals also displayed signs of disease and died within 24 h. TCBS induced 100% mortality in injected starfish. There was no introduction of new pathogens into the marine environment. TCBS promoted the growth of COTS' naturally occurring Vibrionales to high densities with subsequent symbiont imbalance followed by disease and death.

Injection of Acanthaster planci with thiosulfate-citrate-bile-sucrose agar (TCBS). II. Histopathological changes.

We assessed histological changes in the tissues of the crown-of-thorns starfish Acanthaster planci (COTS) after injection of thiosulfate-citrate-bile-sucrose agar (TCBS) which was used as a disease inducer (potential outbreak control method), by conventional and scanning electron microscopy. Digestive glands were processed and stained with hematoxylin and eosin to describe the histological architecture of the intestinal epithelium. Subsequently comparison of healthy versus infected tissues and Gram stains were carried out to confirm bacterial occurrence on infected tissues, characterize the structural changes induced by bacterial communities in COTS tissues, and to determine if the histopathological changes of intestinal tissues were consistent with vibrio infection. TCBS injections induced marked epithelial desquamation, hypertrophy and hypersecretion of glandular cells, epithelial cell destruction, pyknosis, reduction of thickness and disorganization of connective tissue and associated nerve plexus, presence of bacterial colonies, irregular eosinophilic foci in glandular cells, brush border disruption, atrophy and detachment of intestinal microvilli and cell debris in the lumen. All these changes were attributed to a fulminating systemic dysbiosis and were consistent with vibrio infections.