Jairo Rivera-Posada

Lethal doses of oxbile, peptones and thiosulfate-citrate-bile-sucrose agar (TCBS) for Acanthaster planci; exploring alternative population control options.

Effective control of outbreaks of Acanthaster planci represents the most immediate and practical intervention to reverse sustained declines in coral cover on reefs in the Indo-Pacific. This study explored the minimum doses of oxbile, oxgall, and thiosulfate-citrate-bile-sucrose agar (TCBS) that result in reliable and comprehensive mortality when injected into adult A. planci. The minimum doses required to induce 100% mortality among starfish (n=10) were 4 g l(-1) of oxbile, 8 g l(-1) of oxgall and 22 g l(-1) of TCBS. Moreover, there was no evidence of unintended side effects for other coral reef organisms (e.g., scleractinian corals, echinoderms and fishes) when using oxbile, oxgall, or TCBS at minimum doses. The effectiveness of peptones in killing crown-of-thorns starfish was also tested, but inconsistency in the results revealed that these proteins are unreliable.

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.

The Role of Maternal Nutrition on Oocyte Size and Quality, with Respect to Early Larval Development in The Coral-Eating Starfish, Acanthaster planci

Variation in local environmental conditions can have pronounced effects on the population structure and dynamics of marine organisms. Previous studies on crown-of-thorns starfish, Acanthaster planci, have primarily focused on effects of water quality and nutrient availability on larval growth and survival, while the role of maternal nutrition on reproduction and larval development has been overlooked. To examine the effects of maternal nutrition on oocyte size and early larval development in A. planci, we pre-conditioned females for 60 days on alternative diets of preferred coral prey (Acropora abrotanoides) versus non-preferred coral prey (Porites rus) and compared resulting gametes and progeny to those produced by females that were starved over the same period. Females fed ad libitum with Acropora increased in weight, produced heavier gonads and produced larger oocytes compared to Porites-fed and starved females. Fed starfish (regardless of whether it was Acropora or Porites) produced bigger larvae with larger stomachs and had a higher frequency of normal larvae that reached the late bipinnaria / early brachiolaria stage compared to starved starfish. Females on Acropora diet also produced a higher proportion of larvae that progressed to more advanced stages faster compared to Porites-fed starfish, which progressed faster than starved starfish. These results suggest that maternal provisioning can have important consequences for the quality and quantity of progeny. Because food quality (coral community structure) and quantity (coral abundance) varies widely among reef locations and habitats, local variation in maternal nutrition of A. planci is likely to moderate reproductive success and may explain temporal and spatial fluctuations in abundance of this species.

Size-related variation in arm damage frequency in the crown-of-thorns sea star, Acanthaster planci

Objective: To examine variation in the frequency of arm damage in different sizes of Acanthaster planci (A. planci), assess how this damage is inflicted by fish predators, and infer the potential role of predation in population regulation. Methods: Diameters of A. planci collected from three sites in the Philippines were measured and arm damage frequency and severity was assessed. Frequency of arm damage was compared between sizes. Feeding behavior of fish predators was also observed in the laboratory. Results: This study demonstrates that sublethal predation by triggerfishes on A. planci result in extensive arm damage. Overall, 60% of A. planci sampled across all sites had sublethal injuries. The frequency of individuals with missing or regenerating arms was highest in medium-sized young adults (11-20 cm), which coincides with the phase where A. planci shift from cryptic to exposed daytime feeding. Conclusions: The high incidence of arm damage within intermediate-sized sea stars indicates that predators exercise some level of regulation on A. planci populations at a local scale. Identification and protection of putative predators that target the most vulnerable life history stages of A. planci are essential in developing population control strategies and reverse sustained declines in coral cover.

De novo assembly of the transcriptome of Acanthaster planci testes

A key strategy to reduce coral loss is the development of effective control method for the corallivorous crown‐of‐thorns sea star (Acanthaster planci), an omnipresent scourge and threat to the biodiversity of reefs in the Indo‐Pacific region. Limited genetic resources are available for this highly fecund species. In this study, we explored one aspect at the heart of A. planci outbreaks, the male reproductive system. Using high‐throughput sequencing technology, we report for first time the production of a comprehensive transcriptomic data set for the testes of A. placni that can aid in understanding the molecular mechanisms involved in A. planci spermatogenesis and fertilization. Through de novo transcriptome sequencing, we produced 52 965 998 raw reads corresponding to 4.76 Gb clean read data. From this, 243 870 contigs were assembled with Trinity and used to construct 92 792 unigenes. Distinct genes were then annotated with blastx yielding 30 810 unigenes above the cut‐off E‐value set at 10−5, with ESTScan database query analyses yielding up to 5366 unigenes to known hits. The identification of genes directly involved in sperm development (DEAD‐box family proteins), motility, fertilization and signalling (Bindin/Speract receptor) are also discussed.

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.

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.

Osmotic shock as alternative method to control Acanthaster planci

Objective: To test six osmotic stressors as alternative methods to control Acanthaster planci (A. planci) outbreaks by exploiting their incapacity to tolerate drastic changes in osmolarity. Finding more effective ways to control A. planci outbreaks is one of the most immediate and effective ways by which to reverse rapid declines in the abundance of live coral cover in the Indo-Pacific. Methods: A total of 10 mL of each of the following chemicals: sodium chloride, ethylenediaminetetraacetic acid, sodium carbonate, sodium cholate, sodium deoxycholate, urea and mannitol were injected into individual healthy sea stars to examine which chemicals induced disease and death. Results: Four out of six chemicals used in this study induced disease. Sodium chloride, sodium cholate, sodium deoxycholate and ethylenediaminetetraacetic acid are capable of inducing death in injected sea stars offering an alternative option to control A. planci outbreaks. Conclusions: Hyperosmotic stress is a viable alternative to control A. planci outbreaks as massive cell death results when acute hypertonicity exceeds a certain level.

The immune response of Acanthaster planci to oxbile injections and antibiotic treatment

Bile salts have been recently identified as a rapid and effective method for killing A. planci. However the mechanistic basis of this new control method is poorly understood. This study explored the immune response(s) of A. planci and/or pathogenesis resulting from the injection of bile salts. To account for the possible role of pathogenesis in causing high rates of mortality, A. planci was treated with antibiotics to minimise the incidence and severity of bacterial infections. No significant difference in the time to death between groups with and without antibiotic treatment was reported, suggesting a limited bacterial effect on the induction of disease and death of injected sea stars. The number of circulating coelomocytes increased significantly after injection confirming the induction of a strong immune response. Five types of circulating cells were identified: (1) phagocytes, (2) small hyaline cells, (3) colourless spherule cells, (4) red spherule cells, and (5) fusiform cells. Histological analysis of A. planci tissues showed that the mechanism leading to rapid mortality is related to necrosis and/or apoptosis, rather than transmissible disease. Therefore, bile salts are an effective and safe method for killing crown-of-thorns sea star in situ.