Amakusaplana Acroporae, on the Great Barrier Reef, Australia – the First Report from the Wild

Discovery of the Corallivorous Polyclad Flatworm, Amakusaplana acroporae, on the Great Barrier Reef, Australia – the First Report from the Wild Kate A. Rawlinson1*, Jessica S. Stella2,3 1 Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada, 2 Australian Research Council Centre of Excellence for Coral Reef Studies, and School of Marine and Tropical Biology, James Cook University, Townsville, Queensland, Australia, 3 Climate Adaptation Flagship, Commonwealth Scientific and Industrial Research Organisation, Hobart, Tasmania, Australia Abstract The role of corallivory is becoming increasingly recognised as an important factor in coral health at a time when coral reefs around the world face a number of other stressors. The polyclad flatworm, Amakusaplana acroporae, is a voracious predator of Indo-Pacific acroporid corals in captivity, and its inadvertent introduction into aquaria has lead to the death of entire coral colonies. While this flatworm has been a pest to the coral aquaculture community for over a decade, it has only been found in aquaria and has never been described from the wild. Understanding its biology and ecology in its natural environment is crucial for identifying viable biological controls for more successful rearing of Acropora colonies in aquaria, and for our understanding of what biotic interactions are important to coral growth and fitness on reefs. Using morphological, histological and molecular techniques we determine that a polyclad found on Acropora valida from Lizard Island, Australia is A. acroporae. The presence of extracellular Symbiodinium in the gut and parenchyma and spirocysts in the gut indicates that it is a corallivore in the wild. The examination of a size-range of individuals shows maturation of the sexual apparatus and increases in the number of eyes with increased body length. Conservative estimates of abundance show that A. acroporae occurred on 7 of the 10 coral colonies collected, with an average of 2.660.65 (mean 6SE) animals per colony. This represents the first report of A. acroporae in the wild, and sets the stage for future studies of A. acroporae ecology and life history in its natural habitat. Citation: Rawlinson KA, Stella JS (2012) Discovery of the Corallivorous Polyclad Flatworm, Amakusaplana acroporae, on the Great Barrier Reef, Australia – the First Report from the Wild. PLoS ONE 7(8): e42240. doi:10.1371/journal.pone.0042240 Editor: Sebastian C. A. Ferse, Leibniz Center for Tropical Marine Ecology, Germany Received April 11, 2012; Accepted July 5, 2012; Published August 1, 2012 Copyright: ß 2012 Rawlinson, Stella. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Animal collection and field work was funded by the Yugilbar Foundation and Lizard Island Research Station – a facility of the Australian Museum and the Great Barrier Reef Marine Park Authority Science for Management Award awarded to J.S. Stella. Morphological and molecular analysis was funded by the Natural Sciences and Engineering Research Council of Canada grant (A5056) awarded to B.K. Hall. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction been overlooked thus far in most studies of coral-associated animals. One such cryptic polyclad, the Acropora-eating flatworm The role of corallivory on coral reefs is becoming increasingly (commonly known as the AEFW), was recently identified and important to coral reef ecology given the number of other stressors classified as Amakusaplana acroporae Rawlinson et al., 2011 [7]. coral reefs worldwide currently face [1]. Invertebrates are the Known only from aquaria as a notorious pest of Acropora coral, this majority of corallivores, outnumbering their fish counterparts species has never been found in the wild. In fact, the taxonomic nearly 3 to 1 [2,3]. However, most invertebrate species have long assignment was based on multiple specimens collected from two been overlooked due to their small size and cryptic nature [4]. aquaria in the United States. Although most small animals that Corallivorous invertebrates may play an important role in coral live and feed on corals have negligible, if any, ill effects on the health, inflicting minor or lethal damage on their coral hosts, coral host [8], infestations of A. acroporae on acroporids in captivity which may subsequently have deleterious effects on coral growth can result in rapid and complete colony death [9]. A. acroporae is a and fitness [1]. They have also been implicated in transmitting or destructive predator of at least nine aquarium-reared Indo-Pacific increasing vulnerability to coral disease [5], which indirectly acroporids (Acropora valida, A. pulchra, A. millepora, A. tortuosa, A. nana, contributes to coral loss or shifts in community composition. As A. tenuis, A. formosa, A. echinata and A. yongei), individuals lay multiple scleractinian corals are the major reef builders, more attention is egg batches on an Acropora host and the hatchlings have a low required to identify their predators and determine the roles they dispersal capability [7]. These life history characteristics, com- might play in maintaining or conserving coral reef ecosystems. bined with high prey specificity to Acropora, lend this species the Two species of polyclad flatworms are known to prey on potential to be a significant corallivore of Acropora corals. scleractinian corals [6,7], yet very little is known about their Corallivory on Acropora corals is of particular interest to impacts on coral reefs. As they are small and difficult to detect due conservation management as Acropora is one of the most to their excellent camouflage against the coral host, they may have ecologically important coral genera to coral reefs worldwide. It is PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42240 Amakusaplana acroporae on the Great Barrier Reef the largest extant coral genus, occurring in all tropical oceans as sections stained with DAPI (49, 6-Diamidino-2-phenylindole, the dominant reef building coral [10]. Acroporids are a source of Sigma). Five individuals were sectioned in the transverse plane, critical habitat and food for an immense diversity (,150 species) of three individuals were sectioned in the sagittal plane, and two coral-associated animals [3,11,12]. They are extremely abundant individuals mounted as whole mounts. All material, including and fast-growing branching corals yet are among the most whole specimens, has been deposited in the Museum of Tropical susceptible corals to bleaching [13] and disease [14]. Furthermore, Queensland. many corallivores actively select species of Acropora as their preferred prey [1,2], such as the crown-of-thorns sea star, Molecular Analysis Acanthaster planci [15] and the gastropod Drupella conus [16]. Genomic DNA was extracted from one adult specimen Acroporids are also commercially important, being among the (G20079) and the D1–D2 region of the 28 S rDNA gene was top three genera collected for the aquarium trade [17]. Thus amplified using a novel forward (39–59) and reverse (39–59) primer acroporids are often the focus of conservation efforts, such as reef pair designed for Amakusaplana acroporae based on conserved regions restoration [18], and an understanding of what biotic and abiotic within aligned polyclad 28S rDNA sequences [7]. PCR was interactions affect the growth, survival and distribution of carried out using the following cycle temperatures/times: 4 min at acroporid corals is critical to their effective conservation. 94uC; 45 cycles of 20 s at 94uC, 20 s at 52.5uC and 90 s at 72uC; Given Amakusaplana acroporae’s preference for Indo-Pacific 8 min at 72uC for a final extension. PCR was electrophoresed in a Acropora species it is assumed that the worm is endemic to that 1% agarose gel, and the product was excised and purified using region. Its cryptic coloration and relatively small size would make the Qiagen MinElute Gel Extraction kit. The amplified fragment it difficult to detect in situ, hence its easy introduction into aquaria was cloned and sequenced in both directions using the pGem-T as Acropora epifauna. Locating A. acroporae in its natural environ- easy vector system (Promega). The 28 S rDNA D1-D2 region of ment would permit further study of its biology and ecological G20079 (Genbank accession number JQ791553) was aligned using interactions, and this, in turn, could lead to the discovery of the ClustalW algorithm in MacVector with the polyclad sequences effective biological controls for this corallivore in captivity. This used in Rawlinson et al [7] (outgroup Macrostomum lignano). study aimed to determine whether an as-yet unidentified polyclad Phylogenetic trees were constructed using Bayesian Inference flatworm found on Acropora valida colonies from Lizard Island, (BI) in MrBayes 3.2 [19]. The analysis was performed for Australia, was Amakusaplana acroporae. 2,000,000 generations with a sampling frequency of 100. Node support was determined by posterior probabilities. Materials and Methods Results Animal Collection and Fixation Animals were collected from Lizard Island, in the northern Morphological Analysis Great Barrier Reef, Australia (Fig. 1a) (under the

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