Seasonal tissue-specific gene expression in wild crown-of-thorns starfish reveals reproductive and stress-related transcriptional systems.

Animals are influenced by the season, yet we know little about the changes that occur in most species throughout the year. This is particularly true in tropical marine animals that experience relatively small annual temperature and daylight changes. Like many coral reef inhabitants, the crown-of-thorns starfish (COTS), well known as a notorious consumer of corals and destroyer of coral reefs, reproduces exclusively in the summer. By comparing gene expression in 7 somatic tissues procured from wild COTS sampled on the Great Barrier Reef, we identified more than 2,000 protein-coding genes that change significantly between summer and winter. COTS genes that appear to mediate conspecific communication, including both signalling factors released into the surrounding sea water and cell surface receptors, are up-regulated in external secretory and sensory tissues in the summer, often in a sex-specific manner. Sexually dimorphic gene expression appears to be underpinned by sex- and season-specific transcription factors (TFs) and gene regulatory programs. There are over 100 TFs that are seasonally expressed, 87% of which are significantly up-regulated in the summer. Six nuclear receptors are up-regulated in all tissues in the summer, suggesting that systemic seasonal changes are hormonally controlled, as in vertebrates. Unexpectedly, there is a suite of stress-related chaperone proteins and TFs, including HIFa, ATF3, C/EBP, CREB, and NF-κB, that are uniquely and widely co-expressed in gravid females. The up-regulation of these stress proteins in the summer suggests the demands of oogenesis in this highly fecund starfish affects protein stability and turnover in somatic cells. Together, these circannual changes in gene expression provide novel insights into seasonal changes in this coral reef pest and have the potential to identify vulnerabilities for targeted biocontrol.

Potential for host-symbiont communication via neurotransmitters and neuromodulators in an aneural animal, the marine sponge Amphimedon queenslandica.

Interkingdom signalling within a holobiont allows host and symbionts to communicate and to regulate each other's physiological and developmental states. Here we show that a suite of signalling molecules that function as neurotransmitters and neuromodulators in most animals with nervous systems, specifically dopamine and trace amines, are produced exclusively by the bacterial symbionts of the demosponge Amphimedon queenslandica. Although sponges do not possess a nervous system, A. queenslandica expresses rhodopsin class G-protein-coupled receptors that are structurally similar to dopamine and trace amine receptors. When sponge larvae, which express these receptors, are exposed to agonists and antagonists of bilaterian dopamine and trace amine receptors, we observe marked changes in larval phototactic swimming behaviour, consistent with the sponge being competent to recognise and respond to symbiont-derived trace amine signals. These results indicate that monoamines synthesised by bacterial symbionts may be able to influence the physiology of the host sponge.

Captivity induces a sweeping and sustained genomic response in a starfish.

Marine animals in the wild are often difficult to access, so they are studied in captivity. However, the implicit assumption that physiological processes of animals in artificial environments are not different from those in the wild has rarely been tested. Here, we investigate the extent to which an animal is impacted by captivity by comparing global gene expression in wild and captive crown-of-thorns starfish (COTS). In a preliminary analysis, we compared transcriptomes of three external tissues obtained from multiple wild COTS with a single captive COTS maintained in aquaria for at least 1 week. On average, an astonishingly large 24% of the coding sequences in the genome were differentially expressed. This led us to conduct a replicated experiment to test more comprehensively the impact of captivity on gene expression. Specifically, a comparison of 13 wild with 8 captive COTS coelomocyte transcriptomes revealed significant differences in the expression of 20% of coding sequences. Coelomocyte transcriptomes in captive COTS remain different from those in wild COTS for more than 30 days and show no indication of reverting back to a wild state (i.e. no evidence of acclimation). Genes upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in cell signalling. These changes in gene expression indicate that being translocated and maintained in captivity has a marked impact on the physiology and health of these echinoderms. This study suggests that caution should be exercised when extrapolating results from captive aquatic invertebrates to their wild counterparts.

How larvae and life cycles evolve.

Marine larvae have factored heavily in pursuits to understand the origin and evolution of animal life cycles. Recent comparisons of gene expression and chromatin state in different species of sea urchin and annelid show how evolutionary changes in embryonic gene regulation can lead to markedly different larval forms.

Antioxidant enzymes that target hydrogen peroxide are conserved across the animal kingdom, from sponges to mammals.

Oxygen is the sustenance of aerobic life and yet is highly toxic. In early life, antioxidants functioned solely to defend against toxic effects of reactive oxygen species (ROS). Later, as aerobic metabolisms evolved, ROS became essential for signalling. Thus, antioxidants are multifunctional and must detoxify, but also permit ROS signalling for vital cellular processes. Here we conduct metazoan-wide genomic assessments of three enzymatic antioxidant families that target the predominant ROS signaller, hydrogen peroxide: namely, monofunctional catalases (CAT), peroxiredoxins (PRX), and glutathione peroxidases (GPX). We reveal that the two most evolutionary ancient families, CAT and PRX, exhibit metazoan-wide conservation. In the basal animal lineage, sponges (phylum Porifera), we find all three antioxidant families, but with GPX least abundant. Poriferan CATs are distinct from bilaterian CATs, but the evolutionary divergence is small. Amongst PRXs, subfamily PRX6 is the most conserved, whilst subfamily AhpC-PRX1 is the largest; PRX4 is the only core member conserved from sponges to mammals and may represent the ancestral animal AhpC-PRX1. Conversely, for GPX, the most recent family to arise, only the cysteine-dependent subfamily GPX7 is conserved across metazoans, and common across Porifera. Our analyses illustrate that the fundamental functions of antioxidants have resulted in gene conservation throughout the animal kingdom.

Sex-specific expression of pheromones and other signals in gravid starfish.

BACKGROUND: Many echinoderms form seasonal aggregations prior to spawning. In some fecund species, a spawning event can lead to population outbreaks with detrimental ecosystem impacts. For instance, outbreaks of crown-of-thorns starfish (COTS), a corallivore, can destroy coral reefs. Here, we examine the gene expression in gravid male and female COTS prior to spawning in the wild, to identify genome-encoded factors that may regulate aggregation and spawning. This study is informed by a previously identified exoproteome that attracts conspecifics. To capture the natural gene expression profiles, we isolated RNAs from gravid female and male COTS immediately after they were removed from the Great Barrier Reef.  RESULTS: Sexually dimorphic gene expression is present in all seven somatic tissues and organs that we surveyed and in the gonads. Approximately 40% of the exoproteome transcripts are differentially expressed between sexes. Males uniquely upregulate an additional 68 secreted factors in their testes. A suite of neuropeptides in sensory organs, coelomocytes and gonads is differentially expressed between sexes, including the relaxin-like gonad-stimulating peptide and gonadotropin-releasing hormones. Female sensory tentacles-chemosensory organs at the distal tips of the starfish arms-uniquely upregulate diverse receptors and signalling molecules, including chemosensory G-protein-coupled receptors and several neuropeptides, including kisspeptin, SALMFamide and orexin. CONCLUSIONS: Analysis of 103 tissue/organ transcriptomes from 13 wild COTS has revealed genes that are consistently differentially expressed between gravid females and males and that all tissues surveyed are sexually dimorphic at the molecular level. This finding is consistent with female and male COTS using sex-specific pheromones to regulate reproductive aggregations and synchronised spawning events. These pheromones appear to be received primarily by the sensory tentacles, which express a range of receptors and signalling molecules in a sex-specific manner. Furthermore, coelomocytes and gonads differentially express signalling and regulatory factors that control gametogenesis and spawning in other echinoderms.

Distribution and diversity of ROS-generating enzymes across the animal kingdom, with a focus on sponges (Porifera).

BACKGROUND: Reactive derivatives of oxygen (reactive oxygen species; ROS) are essential in signalling networks of all aerobic life. Redox signalling, based on cascades of oxidation-reduction reactions, is an evolutionarily ancient mechanism that uses ROS to regulate an array of vital cellular processes. Hydrogen peroxide (H2O2) and superoxide anion (O2•-) are employed as signalling molecules that alter the oxidation state of atoms, inhibiting or activating gene activity. Here, we conduct metazoan-wide comparative genomic assessments of the two enzyme families, superoxide dismutase (SOD) and NADPH oxidases (NOX), that generate H2O2 and/or O2•- in animals. RESULTS: Using the genomes of 19 metazoan species representing 10 phyla, we expand significantly on previous surveys of these two ancient enzyme families. We find that the diversity and distribution of both the SOD and NOX enzyme families comprise some conserved members but also vary considerably across phyletic animal lineages. For example, there is substantial NOX gene loss in the ctenophore Mnemiopsis leidyi and divergent SOD isoforms in the bilaterians D. melanogaster and C. elegans. We focus particularly on the sponges (phylum Porifera), a sister group to all other metazoans, from which these enzymes have not previously been described. Within Porifera, we find a unique calcium-regulated NOX, the widespread radiation of an atypical member of CuZnSOD named Rsod, and a novel endoplasmic reticulum MnSOD that is prevalent across aquatic metazoans. CONCLUSIONS: Considering the precise, spatiotemporal specificity of redox signalling, our findings highlight the value of expanding redox research across a greater diversity of organisms to better understand the functional roles of these ancient enzymes within a universally important signalling mechanism.

Gene activation of metazoan Fox transcription factors at the onset of metamorphosis in the marine demosponge Amphimedon queenslandica.

Transcription factors encoded by the Forkhead (Fox) gene family have diverse, sometimes conserved, regulatory roles in eumetazoan development, immunity, and physiology. Although this gene family includes members that predate the origin of the animal kingdom, the majority of metazoan Fox genes evolved after the divergence of animals and choanoflagellates. Here, we characterize the composition, structure, and expression of Fox genes in the marine demosponge Amphimedon queenslandica to better understand the origin and evolution of this family. The Fox gene repertoire in A. queenslandica appears to be similar to the ancestral metazoan Fox gene family. All 17 A. queenslandica Fox genes are differentially expressed during development and in adult cell types. Remarkably, eight of these, all of which appear to be metazoan-specific, are induced within just 1 h of larval settlement and commencement of metamorphosis. Gene co-expression analyses suggest that these eight Fox genes regulate developmental and physiological processes similar to their roles in other animals. These findings are consistent with Fox genes playing deeply ancestral roles in animal development and physiology, including in response to changes in the external environment.

Ribosomal RNA-Depletion Provides an Efficient Method for Successful Dual RNA-Seq Expression Profiling of a Marine Sponge Holobiont.

Investigations of host-symbiont interactions can benefit enormously from a complete and reliable holobiont gene expression profiling. The most efficient way to acquire holobiont transcriptomes is to perform RNA-Seq on both host and symbionts simultaneously. However, optimal methods for capturing both host and symbiont mRNAs are still under development, particularly when the host is a eukaryote and the symbionts are bacteria or archaea. Traditionally, poly(A)-enriched libraries have been used to capture eukaryotic mRNA, but the ability of this method to adequately capture bacterial mRNAs is unclear because of the short half-life of the bacterial transcripts. Here, we address this gap in knowledge with the aim of helping others to choose an appropriate RNA-Seq approach for analysis of animal host-bacterial symbiont transcriptomes. Specifically, we compared transcriptome bias, depth and coverage achieved by two different mRNA capture and sequencing strategies applied to the marine demosponge Amphimedon queenslandica holobiont. Annotated genomes of the sponge host and the three most abundant bacterial symbionts, which can comprise up to 95% of the adult microbiome, are available. Importantly, this allows for transcriptomes to be accurately mapped to these genomes, and thus quantitatively assessed and compared. The two strategies that we compare here are (i) poly(A) captured mRNA-Seq (Poly(A)-RNA-Seq) and (ii) ribosomal RNA depleted RNA-Seq (rRNA-depleted-RNA-Seq). For the host sponge, we find no significant difference in transcriptomes generated by the two different mRNA capture methods. However, for the symbiont transcriptomes, we confirm the expectation that the rRNA-depleted-RNA-Seq performs much better than the Poly(A)-RNA-Seq. This comparison demonstrates that RNA-Seq by ribosomal RNA depletion is an effective and reliable method to simultaneously capture gene expression in host and symbionts and thus to analyse holobiont transcriptomes.

Phototransduction in a marine sponge provides insights into the origin of animal vision.

Most organisms respond to light. Here, we investigate the origin of metazoan phototransduction by comparing well-characterized opsin-based photosystems in neural animals with those in the sponge Amphimedon queenslandica. Although sponges lack neurons and opsins, they can respond rapidly to light. In Amphimedon larvae, this is guided by the light-sensing posterior pigment ring. We first use cell-type-specific transcriptomes to reveal that genes that characterize eumetazoan Gt- and Go-mediated photosystems are enriched in the pigment ring. We then apply a suite of signaling pathway agonists and antagonists to swimming larvae exposed to directional light. These experiments implicate metabotropic glutamate receptors, phospholipase-C, protein kinase C, and voltage-gated calcium channels in larval phototaxis; the inhibition of phospholipase-C, a key transducer of the Gq-mediated pathway, completely reverses phototactic behavior. Together, these results are consistent with aneural sponges sharing with neural metazoans an ancestral set of photosignaling pathways.

Distal regulation, silencers, and a shared combinatorial syntax are hallmarks of animal embryogenesis.

The chromatin environment plays a central role in regulating developmental gene expression in metazoans. Yet, the ancestral regulatory landscape of metazoan embryogenesis is unknown. Here, we generate chromatin accessibility profiles for six embryonic, plus larval and adult stages in the sponge Amphimedon queenslandica These profiles are reproducible within stages, reflect histone modifications, and identify transcription factor (TF) binding sequence motifs predictive of cis-regulatory elements operating during embryogenesis in other metazoans, but not the unicellular relative Capsaspora Motif analysis of chromatin accessibility profiles across Amphimedon embryogenesis identifies three major developmental periods. As in bilaterian embryogenesis, early development in Amphimedon involves activating and repressive chromatin in regions both proximal and distal to transcription start sites. Transcriptionally repressive elements ("silencers") are prominent during late embryogenesis. They coincide with an increase in cis-regulatory regions harboring metazoan TF binding motifs, as well as an increase in the expression of metazoan-specific genes. Changes in chromatin state and gene expression in Amphimedon suggest the conservation of distal enhancers, dynamically silenced chromatin, and TF-DNA binding specificity in animal embryogenesis.

Arginine Biosynthesis by a Bacterial Symbiont Enables Nitric Oxide Production and Facilitates Larval Settlement in the Marine-Sponge Host.

Larval settlement and metamorphosis are regulated by nitric oxide (NO) signaling in a wide diversity of marine invertebrates.1-10 It is thus surprising that, in most invertebrates, the substrate for NO synthesis-arginine-cannot be biosynthesized but instead must be exogenously sourced.11 In the sponge Amphimedon queenslandica, vertically inherited proteobacterial symbionts in the larva are able to biosynthesize arginine.12,13 Here, we test the hypothesis that symbionts provide arginine to the sponge host so that nitric oxide synthase expressed in the larva can produce NO, which regulates metamorphosis,8 and the byproduct citrulline (Figure 1). First, we find support for an arginine-citrulline biosynthetic loop in this sponge larval holobiont by using stable isotope tracing. In symbionts, incorporated 13C-citrulline decreases as 13C-arginine increases, consistent with the use of exogenous citrulline for arginine synthesis. In contrast, 13C-citrulline accumulates in larvae as 13C-arginine decreases, demonstrating the uptake of exogenous arginine and its conversion to NO and citrulline. Second, we show that, although Amphimedon larvae can derive arginine directly from seawater, normal settlement and metamorphosis can occur in artificial sea water lacking arginine. Together, these results support holobiont complementation of the arginine-citrulline loop and NO biosynthesis in Amphimedon larvae, suggesting a critical role for bacterial symbionts in the development of this marine sponge. Given that NO regulates settlement and metamorphosis in diverse animal phyla1-10 and arginine is procured externally in most animals,11 we propose that symbionts might play an equally critical regulatory role in this essential life cycle transition in other metazoans.

Staining and Tracking Methods for Studying Sponge Cell Dynamics.

To better understand the origin of animal cell types, body plans, and other morphological features, further biological knowledge and understanding are needed from non-bilaterian phyla, namely, Placozoa, Ctenophora, and Porifera. This chapter describes recent cell staining approaches that have been developed in three phylogenetically distinct sponge species-the homoscleromorph Oscarella lobularis, and the demosponges Amphimedon queenslandica and Lycopodina hypogea-to enable analyses of cell death, proliferation, and migration. These methods allow for a more detailed understanding of cellular behaviors and fates, and morphogenetic processes in poriferans, building on current knowledge of sponge cell biology that relies chiefly on classical (static) histological observations.

Molecular and behavioural evidence that interdependent photo - and chemosensory systems regulate larval settlement in a marine sponge.

Marine pelagic larvae use a hierarchy of environmental cues to identify a suitable benthic habitat on which to settle and metamorphose into the adult phase of the life cycle. Most larvae are induced to settle by biochemical cues and many species have long been known to preferentially settle in the dark. Combined, these data suggest that larval responses to light and biochemical cues may be linked, but this has yet to be explored at the molecular level. Here, we track the vertical position of larvae of the sponge Amphimedon queenslandica to show that they descend to the benthos at twilight, by which time they are competent to respond to biochemical cues, consistent with them naturally settling in the dark. We use larval settlement assays under three different light regimes, combined with transcriptomics on individual larvae, to identify candidate molecular pathways underlying larval settlement. We find that larvae do not settle in response to biochemical cues if maintained in constant light. Our transcriptome data suggest that constant light actively represses settlement via the sustained up-regulation of two putative inactivators of chemotransduction in constant light only. Our data suggest that photo- and chemosensory systems interact to regulate larval settlement via nitric oxide and cyclic guanosine monophosphate signalling in this sponge, which belongs to one of the earliest-branching animal phyla.

Co-expression of synaptic genes in the sponge Amphimedon queenslandica uncovers ancient neural submodules.

The synapse is a complex cellular module crucial to the functioning of neurons. It evolved largely through the exaptation of pre-existing smaller submodules, each of which are comprised of ancient sets of proteins that are conserved in modern animals and other eukaryotes. Although these ancient submodules themselves have non-neural roles, it has been hypothesized that they may mediate environmental sensing behaviors in aneural animals, such as sponges. Here we identify orthologues in the sponge Amphimedon queenslandica of genes encoding synaptic submodules in neural animals, and analyse their cell-type specific and developmental expression to determine their potential to be co-regulated. We find that genes comprising certain synaptic submodules, including those involved in vesicle trafficking, calcium-regulation and scaffolding of postsynaptic receptor clusters, are co-expressed in adult choanocytes and during metamorphosis. Although these submodules may contribute to sensory roles in this cell type and this life cycle stage, total synaptic gene co-expression profiles do not support the existence of a functional synapse in A. queenslandica. The lack of evidence for the co-regulation of genes necessary for pre- and post-synaptic functioning in A. queenslandica suggests that sponges, and perhaps the last common ancestor of sponges and other extant animals, had the ability to promulgate sensory inputs without complete synapse-like functionalities. The differential co-expression of multiple synaptic submodule genes in sponge choanocytes, which have sensory and feeding roles, however, is consistent with the metazoan ancestor minimally being able to undergo exo- and endocytosis in a controlled and localized manner.

Author Correction: The mid-developmental transition and the evolution of animal body plans.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Convergent evolution of a vertebrate-like methylome in a marine sponge.

Vertebrates have highly methylated genomes at CpG positions, whereas invertebrates have sparsely methylated genomes. This increase in methylation content is considered a major regulatory innovation of vertebrate genomes. However, here we report that a sponge, proposed as the potential sister group to the rest of animals, has a highly methylated genome. Despite major differences in genome size and architecture, we find similarities between the independent acquisitions of the hypermethylated state. Both lineages show genome-wide CpG depletion, conserved strong transcription factor methyl-sensitivity and developmental methylation dynamics at 5-hydroxymethylcytosine enriched regions. Together, our findings trace back patterns associated with DNA methylation in vertebrates to the early steps of animal evolution. Thus, the sponge methylome challenges previous hypotheses concerning the uniqueness of vertebrate genome hypermethylation and its implications for regulatory complexity.

Pluripotency and the origin of animal multicellularity.

A widely held-but rarely tested-hypothesis for the origin of animals is that they evolved from a unicellular ancestor, with an apical cilium surrounded by a microvillar collar, that structurally resembled modern sponge choanocytes and choanoflagellates1-4. Here we test this view of animal origins by comparing the transcriptomes, fates and behaviours of the three primary sponge cell types-choanocytes, pluripotent mesenchymal archaeocytes and epithelial pinacocytes-with choanoflagellates and other unicellular holozoans. Unexpectedly, we find that the transcriptome of sponge choanocytes is the least similar to the transcriptomes of choanoflagellates and is significantly enriched in genes unique to either animals or sponges alone. By contrast, pluripotent archaeocytes upregulate genes that control cell proliferation and gene expression, as in other metazoan stem cells and in the proliferating stages of two unicellular holozoans, including a colonial choanoflagellate. Choanocytes in the sponge Amphimedon queenslandica exist in a transient metastable state and readily transdifferentiate into archaeocytes, which can differentiate into a range of other cell types. These sponge cell-type conversions are similar to the temporal cell-state changes that occur in unicellular holozoans5. Together, these analyses argue against homology of sponge choanocytes and choanoflagellates, and the view that the first multicellular animals were simple balls of cells with limited capacity to differentiate. Instead, our results are consistent with the first animal cell being able to transition between multiple states in a manner similar to modern transdifferentiating and stem cells.

The first identification of complete Eph-ephrin signalling in ctenophores and sponges reveals a role for neofunctionalization in the emergence of signalling domains.

BACKGROUND: Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores - non-bilaterian basal animals - typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected. RESULTS: Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported. CONCLUSIONS: We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.