Zoomorphology (2011) 130:261–271 DOI 10.1007/s00435-011-0135-y ORIGINAL PAPER Microanatomy of the trophosome region of Paracatenula cf. polyhymnia (Catenulida, Platyhelminthes) and its intracellular symbionts Nikolaus Leisch • Ulrich Dirks • Harald R. Gruber-Vodicka • Markus Schmid • Wolfgang Sterrer • Jo¨rg A. Ott Received: 13 June 2011 / Revised: 11 August 2011 / Accepted: 13 August 2011 / Published online: 14 September 2011 Ó The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Marine catenulid platyhelminths of the genus morphologically reduced and most likely not functional. Paracatenula lack mouth, pharynx and gut. They live in a Cells containing needle-like inclusions in the reference symbiosis with intracellular bacteria which are restricted to species Paracatenula polyhymnia Sterrer and Rieger, 1974 the body region posterior to the brain. The symbiont- were thought to be sperm, and the inclusions interpreted as housing cells (bacteriocytes) collectively form the tropho- the sperm nucleus. Our analysis of similar cells and their some tissue, which functionally replaces the digestive tract. inclusions by EDX and Raman microspectroscopy docu- It constitutes the largest part of the body and is the most ments an inorganic spicule consisting of a unique magne- important synapomorphy of this group. While some other sium–phosphate compound. Furthermore, we identify the features of the Paracatenula anatomy have already been neoblast stem cells located underneath the epidermis. analyzed, an in-depth analysis of the trophosome region Except for the modifications due to the symbiotic lifestyle was missing. Here, we identify and characterize the com- and the enigmatic spicule cells, the organization of Para- position of the trophosome and its surrounding tissue by catenula cf. polyhymnia conforms to that of the Catenulida analyzing series of ultra-thin cross-sections of the species in all studied aspects. Therefore, this species represents an Paracatenula cf. polyhymnia. For the first time, a proto- excellent model system for further studies of host adapta- nephridium is detected in a Paracatenula species, but it is tion to an obligate symbiotic lifestyle. Keywords Platyhelminthes Á Symbiosis Á Paracatenula Á Ultrastructure Á Trophosome Communicated by T. Bartolomaeus. Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00435-011-0135-y) contains supplementary Morphological and molecular evidence indicates that the material, which is available to authorized users. Catenulida are the most basal group of Platyhelminthes (Ehlers 1985; Larsson and Jondelius 2008; Philippe et al. N. Leisch (&) Á U. Dirks Á H. R. Gruber-Vodicka Á J. A. Ott 2011). Most species of the order are limnic, but the Ret- Department of Marine Biology, University of Vienna, ronectidae were initially reported to be exclusively marine Althanstraße 14, 1090 Vienna, Austria (Sterrer and Rieger 1974) and containing two genera, e-mail: [email protected] Retronectes and Paracatenula. Noren˜a and Faubel (1996) M. Schmid described a third genus—Myoretronectes from a limnic Department of Microbial Ecology, University of Vienna, habitat. While Retronectes species and most limnic Cate- Althanstraße 14, 1090 Vienna, Austria nulida have been reported from tropical to cold temperate regions, the members of the genus Paracatenula have been W. Sterrer Bermuda Aquarium, Natural History Museum and Zoo (BAMZ), found only in warm-temperate to tropical regions. All Flatts Village, Bermuda species of the genus Paracatenula lack mouth, pharynx and 123 262 Zoomorphology (2011) 130:261–271 Fig. 1 Habitus of Paracatenula cf. polyhymnia. a Light microscope micrograph, dotted rectangle represents area of serial section. b Phase contrast micrograph of the anterior region. tr trophosome region, ro rostrum, sp spicule, ba bacteria gut (Sterrer and Rieger 1974). Instead, the largest part of likely satisfy most or even all of the nutritional demands of the body is made up of a tissue composed of bacteriocytes, the host. cells harboring intracellular bacterial symbionts (Ott et al. Platyhelminthes are known for their unique system of 1982). This tissue is called the trophosome (Dirks et al. totipotent stem cells—the neoblasts—from which all cells 2011), in functional analogy to the trophosome found in the arise and which are the only cells showing mitotic activity mouthless Siboglinidae (Annelida) (Fig. 1). All species of (Ladurner et al. 2000; Peter et al. 2004). On the ultra- the genus Paracatenula inhabit the interstitial space of structural level, they can be identified by the following shallow-water subtidal sands. In this habitat, chemoauto- structures: (1) a highly active nucleus displaying a check- trophic primary production by prokaryotes oxidizing erboard pattern, (2) many mitochondria, (3) free ribosomes reduced sulfur compounds plays a vital role comparable to and (4) chromatoid bodies (Hori 1982). The chromatoid hydrothermal hot vents, cold seeps and whale- and wood- bodies are electron-dense structures that can be found close falls. Mutualistic symbioses with sulfur-oxidizing bacteria to the nuclear envelope and mitochondria. They contain have been documented from these habitats for many taxa of RNA and proteins and are essential for the cytodifferenti- eukaryotes, including ciliates, nematodes, mollusks and ation process (Auladell et al. 1993). The ultrastructural annelids. Most of these show profound morphological investigation of the neoblast also gave the opportunity to adaptations to the symbiotic lifestyle, such as reduction in add to the knowledge of the anatomy of this exceptional mouth and gut, specialized symbiont-housing tissues and representative of the Catenulids, a group that is less well reduction in excretory organs. The symbionts belong to studied than many other platyhelminth taxa (Bedini and several different clades in the Alpha-, Gamma-orEpsi- Papi 1974; Ehlers 1985; Hooge 2001; Moraczweski 1981; lonproteobacteria (Dubilier et al. 2008; Gruber-Vodicka Rieger et al. 1991; Rohde 2001). et al. 2011). The association between Paracatenula species While several studied features of Paracatenula, such as and the thiotrophic Alphaproteobacteria Candidatus Rie- muscle arrangement (Hooge 2001) or brain organization geria likely dates back to the early days of platyhelminth (Sterrer and Rieger 1974), are typical for Catenulida, the evolution more than 500 million years ago (Gruber-Vodi- synapomorphy of this genus is the lack of a digestive tract cka et al. 2011). Similar to what has been shown for other and its functional replacement by the trophosome tissue. meiofaunal hosts of sulfur-oxidizing bacteria, e.g., nema- We therefore focused on this region in Paracatenula cf. todes (Ott et al. 1991) or gutless oligochaetes (Giere et al. polyhymnia, using serial-sectioning-based transmission 1991), the animals probably migrate through the redox electron microscopic reconstructions, scanning electron potential gradient in the top 5–15 cm layer of the sediment microscopy, together with energy dispersive X-ray analysis and thus could provide their symbionts with both sulfide and Raman microspectroscopy techniques. We character- and electron acceptors such as nitrate or oxygen. In ized the tissue composition and resolved enigmatic struc- endosymbiotic associations, such as in the genus Paraca- tures that are unique to these worms, such as the spicule- tenula, the host additionally provides a sheltered and stable shaped inclusions, previously regarded as sperm, which are environment to its symbionts. In return, the symbionts in fact of mineral nature (‘‘sp’’ in Fig. 1b). 123 Zoomorphology (2011) 130:261–271 263 Materials and methods Genesis software (Ver. 5.11 EDAX). Readings from the carbon-coated stub served as a signal background for our Specimens were collected during two field trips (October measurements. For Raman microspectroscopy, only indi- 2009, June 2010) in Dahab, Egypt. Sampling took place in viduals that had not been relaxed with magnesium chloride ‘‘Eel Garden’’ (28°30019.9700N, 34°31018.5200E; 8 m depth) were used. Both semi-thin sections (2.5 lm) of resin- and ‘‘Napoleon Reef’’ (28°28013.8300N, 34°30032.5100E; 1 m embedded specimens and complete spicules were mounted depth). Specimens were extracted from the fine to medium on a calcium fluoride slide, and analysis was performed sand by shaking it with filtered seawater and decanting the with a LabRAM HR800 confocal Raman microscope supernatant through a 32-lm mesh sieve. The content of the (Horiba Jobin–Yvon, Bensheim, Germany), utilizing a net was transferred into a petri dish, and approximately 200 532-nm Nd: YAG laser (50mW). Spicules were selected specimens were sorted by morphological features under a using a 509 objective, and the signal was acquired over a dissecting microscope. Paracatenula cf. polyhymnia was period of 5 s using a D0.6 intensity filter (25% laser identified by the presence of spicules similar to those of intensity). The pinhole of the Peltier-cooled CCD detector Paracatenula polyhymnia, reported from the Western was adjusted to 250 lm (optical slice 4.6 lm). Spectra Atlantic (Sterrer and Rieger 1974). Reference material for were measured between 300 and 2,000 cm-1, the range P. cf. polyhymnia has been deposited in the Natural History where the compounds identified with the EDX showed the Museum Vienna under the numbers NHM Wien/Vienna Ev most characteristic peaks. The spectra were acquired, varia 20.109 and NHM