Reference: Biol. Bull. 236: 66–73. (February 2019) © 2018 The University of Chicago DOI: 10.1086/700834 A New Species of Orthonectida That Parasitizes Xenoturbella bocki: Implications for Studies on Xenoturbella HIROAKI NAKANO* AND HIDEYUKI MIYAZAWA† Shimoda Marine Research Center, University of Tsukuba, 5-10-1, Shimoda, Shizuoka 415-0025, Japan Abstract. Orthonectida is a phylum of marine invertebrates Introduction known to parasitize many invertebrate animals. Because of its Orthonectids, with about 30 described species, are micro- simple body plan, it was suggested that it belong to Mesozoa, scopic animals known as parasites in various marine inver- together with Dicyemida, and that it represent the evolution- tebrates, possessing a peculiar life cycle (Kozloff, 1992, 1993). ary step between unicellular organisms and multicellular an- They have been reported to parasitize acoels, platyhelminthes, imals. Recent studies, including analyses of its genomes, have annelids, molluscs, nemerteans, echinoderms, and chordates; clarified its phylogenetic position as a member of the Protos- and it has been suggested that they are present in the host ani- tomia, but details such as the species diversity within the phy- mals as multinucleate plasmodia (Slyusarev and Miller, 1998). lum and how it infects the host remain unknown. Here we However, the plasmodia may also be a mass of host cells that report orthonectids discovered from the marine worm Xeno- has degenerated due to the parasites, and therefore not a viable turbella bocki. Orthonectids were found from sections of four stage of the orthonectid life cycle (Kozloff, 1994, 1997). Male xenoturbellid specimens, collected eight years apart. Live fe- and female adults are produced from germinal cells within the males were also discovered on three separate occasions. These plasmodia, and the ciliated adults swim out from the hosts. The recurring instances of orthonectids found from Xenoturbella adults have a very simple morphology lacking digestive, excre- show that they are parasitic to the animal and not just chance tory, or circulatory organs. Muscular and nervous systems have contaminations. Based on morphological characters such as previously been reported to be absent; but the presence of both the presence of sexual dimorphism, the arrangement of oocytes structures, although very reduced, has been confirmed (Koz- within the female body, and the presence of crystalline inclu- loff, 1969, 1971; Slyusarev and Starunov, 2016). Instead of sions in the male epidermal cells, we regard this orthonectid these organs, the orthonectid adult body is full of gametes. as a new species, Rhopalura xenoturbellae sp. nov. Since or- Mating results in internal fertilization, and the ciliated larva thonectids are present within the xenoturbellid adult body, cau- swim out of the female and search for new hosts (Caullery tion is needed when interpreting morphological, molecular, and and Lavallee, 1908, 1912; Atkins, 1933). Once inside the host, experimental data from X. bocki. Further studies on R. xeno- the larva disintegrates and forms new plasmodia. However, the turbellae will yield important information on the fundamental complete life cycle of a single species remains to be observed; biological details of orthonectids that remain unknown. how they find and enter the hosts, oogenesis, spermatogenesis, and mating all remain poorly studied. Research on other details of orthonectids, such as diversity within the phylum, physiol- ogy, and nutrient uptake, have also been scarce. Received 28 March 2018; Accepted 16 September 2018; Published online 4 December 2018. Owing to their simple body plan, orthonectids were grouped * To whom correspondence should be addressed. Email: h.nakano@ together with Dicyemida, microscopic parasites found in ceph- shimoda.tsukuba.ac.jp. alopods, in a clade called Mesozoa, a sister group to Metazoa. † Present address: Center for Genome Informatics, Joint Support-Center However, recent studies suggest that the two groups are not for Data Science Research, Research Organization of Information and Sys- basal animals but bilaterians that have secondarily simplified tems, Mishima, Shizuoka 411-8540, Japan. Abbreviations: SMNH, Swedish Museum of Natural History; SMRC, as a result of their parasitic lifestyle. The spiral cleavage, mus- Shimoda Marine Research Center. culature (Slyusarev and Starunov, 2016), and fine structure of Online enhancements: videos. ciliated cells and ciliary rootlets (Slyusarev and Kristensen, 66 This content downloaded from 133.051.140.014 on April 11, 2019 19:08:23 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). ORTHONECTID INFECTING XENOTURBELLA 67 2003) of Orthonectida show similarities to those of annelids; and are not just chance contaminations. Based on the host or- therefore, it has been suggested that they be a member of the ganism and morphological features, we regard the newly Protostomia. Molecular phylogenetic analyses have been in ac- found orthonectid as a new species, Rhopalura xenoturbellae cord with this view (Hanelt et al., 1996; Pawlowski et al., 1996; sp. nov. We consider its probable effect on the reproduction Petrov et al., 2010), with genomic data of Intoshia linei strongly of its host and discuss the parasite’s importance for studies supporting this affinity (Mikhailov et al., 2016). Studies using on X. bocki. Many fundamental biological details that remain transcriptomic data of a dicyemid, Dicyema japonicum,have unknown for orthonectids will be revealed through research also suggested that orthonectids are protostomes (Lu et al., on various species, including this species. 2017); and another analysis on mitochondrial genomes and nuclear genes suggested that orthonectids are members of the Materials and Methods phylum Annelida (Schiffer et al., 2018). Xenoturbella, with six reported species, is another animal Xenoturbella specimens were collected in Gullmarsfjord, with a simple body plan (Nakano, 2015; Rouse et al., 2016; Sweden (approx. 587170N, 117310E), at 80–150-m depth, using Nakano et al., 2017). Although not parasitic, sessile, or micro- aWaren’s dredge and the research vessel Oscar von Sydow, scopic, the animal lacks a centralized nervous system, anus, Sven Loven Centre for Marine Sciences-Kristineberg, Uni- coeloms, and reproductive organs. Because of the simple mor- versity of Gothenburg. The collection dates and the number phology, its phylogenetic position has long remained unde- of collected xenoturbellids are shown in Table 1. The collected cided. Recent molecular phylogenetic analyses mostly agree xenoturbellids were kept in laboratories at 4–10 7C in either of that it forms the clade Xenacoelomorpha together with the the following methods: (1) closed system: animals were kept in Acoelomorpha (Hejnol et al., 2009; Philippe et al., 2011; Can- closed 50-mL tubes (about 115 mm in length and 30 mm in di- non et al., 2016; Robertson et al., 2017). Analyses of their ameter) with natural seawater, without any flow-through water, mitochondrial genome suggest a deuterostome affinity of the or (2) flow-through system: animals were placed in containers clade (Philippe et al., 2011; Robertson et al., 2017), but large- with a mesh (100-mm pore size) placed on both the inflow and scale phylogenomic studies suggest that it is a sister group to outflow tubes, with constant flow of natural seawater. Details all other extant bilaterians (Nephrozoa) (Hejnol et al., 2009; on the collection and rearing methods are described in Nakano Cannon et al., 2016). Therefore, Xenacoelomorpha and Xeno- et al. (2013) and Nakano (2015). All individuals were identi- turbella will contribute to the studies of early bilaterian or deu- fied as Xenoturbella bocki based on their morphology. Xeno- terostome evolution. However, mainly because of the diffi- turbellid specimens were fixed in 4% paraformaldehyde in culties in collecting the animals, research on Xenoturbella has filtered seawater for 6–16hat47C, washed, and paraffinsec- been lagging (Nakano, 2015). For example, except for reports tioned (6 mm), according to Stach et al. (2005). The sections on cleavage stage embryos and swimming hatchlings (Nakano were stained with hematoxylin and eosin. et al., 2013; Nakano, 2015), the complete life cycle of Xeno- Photographs and movies of live orthonectid specimens and turbella is still a mystery, despite its breeding season being first sections of a xenoturbellid collected on March 6, 2006, were reported nearly 70 years ago (Westblad, 1949). taken with a Canon S40 color camera (Tokyo, Japan) on Leica Here we report orthonectids discovered from the marine DMRBE or MZFLIII microscopes (Wetzlar, Germany). worm Xenoturbella bocki Westblad, 1949. They were found Newly collected live orthonectid specimens were fixed in from sections of four specimens, three of which were col- 4% paraformaldehyde in filtered seawater or 2.5% glutaralde- lected and fixed in April 1998 and the other in March 2006. hyde in 0.1 mol L21 sodium cacodylate buffer (pH 7.2) over- Live female orthonectids were observed in May and Novem- night, washed, and kept in 70% ethanol. ber 2006 and in March 2008. These recurring instances of Three Xenoturbella specimens deposited in the Swedish orthonectids suggest that they are parasites of Xenoturbella Museum of Natural History (SMNH) were observed at the Table 1 Occurrences of Rhopalura xenoturbellae sp. nov. from Xenoturbella bocki R. xenoturbellae Date live R. xenoturbellae found Date host Xenoturbella