Author's personal copy Zoologischer Anzeiger 251 (2012) 270–278 Contents lists available at SciVerse ScienceDirect Zoologischer Anzeiger j ournal homepage: www.elsevier.de/jcz The ultrastructure of the mastax of Filinia longiseta (Flosculariaceae, Rotifera): Informational value of the trophi structure and mastax musculature ∗ Diana Wulfken , Wilko H. Ahlrichs Systematics and Evolutionary Biology, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, 26111 Oldenburg, Germany a r t i c l e i n f o a b s t r a c t Article history: The study contributes to the discussion of mastax evolution within Rotifera by giving an insight into the Received 11 November 2011 ultrastructure of the mastax in the rotifer species Filinia longiseta (Flosculariacea) and additionally into the Received in revised form 9 February 2012 bdelloid rotifer species Adineta vaga and Zelinkiella synaptae. The existence of cuticularized jaw elements Accepted 13 February 2012 (trophi) in the mastax, a muscular pharynx, is one of the defining rotiferan characters and the basis Corresponding Editor: Sorensen. on which the monophyletic taxon Gnathifera Ahlrichs 1995a, comprising Rotifera, Gnathostomulida, Micrognathozoa and Acanthocephala, was erected. By means of SEM observations of the trophi and Keywords: ultrathin serial sections (TEM) of the mastax, the internal and external organization of the jaw elements of Cuticular tubes Cuticular rods F. longiseta is reconstructed. TEM sections of the incus of Filinia demonstrate that the fulcrum and the rami Fulcrum are built up by multitudes of tiny cuticular tubes. While tubular substructures in the rotiferan fulcrum Manubrium have been described previously, distinct cuticular tubes as a substructure of the ramus have only been Ramus described for species belonging to the taxa Seisonidea and Bdelloidea so far (Koehler and Hayes, 1969; Ahlrichs, 1995b). By comparing the appearance and arrangement of the cuticular tubes in the rami of F. longiseta to those found in species of Seisonidea and Bdelloidea, a higher degree of resemblance between the structures in F. longiseta and Bdelloidea can be reported. The occurrence of the ramus substructures in species of Seisonidea (Paraseison annulatus and Seison nebaliae) is given consideration to represent an intermediate between the ramus substructure of Bdelloidea/Flosculariacea and Ploima. Additionally, the mastax musculature of F. longiseta, being associated with the trophi, is described: A total of seven muscles are found that directly insert the jaw elements or are indirectly associated with them via muscle- to-muscle connections. © 2012 Elsevier GmbH. All rights reserved. 1. Introduction The basic arrangement of jaw elements is modified across differ- ent families and species, reflecting their modes of life and feeding The Rotifera traditionally comprise cosmopolitan aquatic strategies. According to the diverging morphology and function of micrometazoans with body lengths of up to one millimeter, in rare the trophi, different basic mastax types can be distinguished (mal- cases even larger. To date, about 2000 species have been described leate, modified malleate, ramate, malleoramate, fulcrate, incudate, (Wallace et al., 2006; Segers, 2007), most of which live in freshwater cardate, uncinate, virgate and forcipate; compare De Beauchamp, environments but also in marine waters and semiaquatic habitats 1909; Remane, 1929–1933; Wallace et al., 2006). Within Rotifera such as damp mosses. Recent molecular studies (see Sørensen and the trophi are used for species identification and are one of the most Giribet, 2006 and Garcia-Varela and Nadler, 2006) also recognize important characters in rotifer systematics and phylogeny. the parasitic Acanthocephala as a rotiferan class. Based on the pharyngeal jaw elements existing in the One of the most conspicuous rotiferan characters is the muscular mastax, Ahlrichs (1995a) erected the monophyletic taxon pharynx, referred to as the mastax, which contains a set of cuticu- Gnathifera, including Rotifera, Gnathostomulida, Limnognathia larized jaw elements (trophi). The basic trophi set comprises paired maerski (Micrognathozoa), and the jaw-less Acanthocephala (for manubria and unci (together referred to as malleus), an unpaired which a close relationship to Rotifera has been suggested, mainly fulcrum and paired rami (together referred to as incus). Specific based on a similar ultrastructure of the syncytial epidermis). muscles serve to move these elements against one thereby allowing In a phylogenetic analysis of rotifer families primarily based on the trophi to penetrate, crush and/or scrape food items. morphological characters of the trophi elements, Sørensen (2002) presents a strict consensus tree in which the large rotifer taxon Monogononta splits into Ploima and Gnesiotrocha (Flosculariacea ∗ and Collothecacea) (Fig. 1A). While the different families of Ploima Corresponding author. E-mail address: [email protected] (D. Wulfken). comprise species with malleate, cardate, virgate, incudate, and 0044-5231/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2012.02.001 Author's personal copy D. Wulfken, W.H. Ahlrichs / Zoologischer Anzeiger 251 (2012) 270–278 271 Fig. 1. Phylogenetic relationships within Rotifera and the species Filinia longiseta (Flosculariacea). (A) Strict consensus tree of a maximum parsimony analysis of Rotifera based on morphological characters according to Sørensen (2002). Seisonidea, Bdelloidea and Monogononta together form the phylum Rotifera; (B) the rotifer species Filinia longiseta (Flosculariacea) and its digestive tract (modified after Sanoamuang, 1993). ma, mastax; mo, mouth opening; oe, oesophagus; sto, stomach; tr, trophi. forcipate mastax types, flosculariacean species display only one Individual specimens were isolated from samples under a stere- type: the malleoramate mastax. The trophi of Filinia longiseta, omicroscope and studied by differential interference contrast light which is the flosculariacean focused on in this study, are charac- microscopy (Leica DMLB) and transmission electron microscopy terized, among others, by a multitude of differentiated unci teeth, (Zeiss 902 TEM). The isolated trophi were examined under a scan- a short fulcrum and the lack of a manubrial cauda (Fig. 2A–D). ning electron microscope (Zeiss DSM 940 and Hitachi S-3200N). With regard to several morphological similarities of the trophi For scanning electron microscopy (SEM) living specimens (sickle-shaped manubria; differentiated unci teeth; see Fig. 2A–D), were treated with SDS/DTT (SDS = sodium dodecyl sulfate, the jaw elements of Flosculariacea and Bdelloidea, the latter being DTT = dithiothreitol) following the protocol given by Kleinow et al. an outgroup to Monogononta sensu Sørensen (2002) (Fig. 1A), were (1990) to dissolve the body and to isolate the trophi. Trophi were compared in several studies before (Markevich, 1985, 1989; Melone rinsed with distilled water and afterwards pipetted onto a stub. et al., 1998). After air-drying, the material was sputter coated with platinum In this study, we contribute to the discussion of mastax and then examined by SEM. evolution within Rotifera by providing detailed insights into For transmission electron microscopy (TEM), specimens were the ultrastructure of the mastax of species from three lineages: anesthetized with carbonated water and then fixed with 1% OsO4 ◦ F. longiseta (Flosculariacea), Paraseison annulatus (Seisonidea), buffered in 0.1 M sodiumcacodylate at 4 C for 1 h. Afterwards, Adineta vaga and Zelinkiella synaptae (Bdelloidea). While the mas- specimens were dehydrated through an increasing acetone series, ◦ tax of species from Seisonidea and Bdelloidea have been the subject embedded in araldite and hardened at 60 C for 72 h. Ultrathin of former TEM-studies (Koehler and Hayes, 1969; Ahlrichs, 1995b), serial sections (horizontal- and cross-sections) of 80 nm of two our analysis focuses on the malleoramate mastax of F. longiseta, female individuals for each species were made with a Reichert ◦ which never was the subject of an ultrastructural study before. Ultracut followed by automatic staining with uranyl acetate (25 C ◦ Filinia longiseta (Fig. 1B) reaches body-lengths of 130–250 ␮m for 25 min) and lead citrate (30 C for 30 min) in a Leica EM Stain. (Sanoamuang, 2002) and can be found in lakes and ponds where it Stained sections were examined and photographed in a TEM at often occurs in large numbers during warmer seasons. The genus 80 kV. Filinia feeds upon small algae, flagellates, bacteria and detritus. The reconstruction of the trophi is based on observations made Their trophi, reaching lengths of 24–30 ␮m (Nogrady and Segers, by means of SEM and TEM: Complete series of cross- and hor- 2002), perform a constant opening and closing movement during izontal sections through the mastax region of specimens were ingestion. photographed at regular intervals using the MIA (multiple image ® alignment) function of iTEM software. The composites show larger structures at higher magnifications and better resolution 2. Material and methods than would be obtained from single images. Serial sections were examined and compared to SEM photographs to obtain informa- All individuals of F. longiseta were collected in a private pond in tion on the three-dimensional structure of the trophi. Line drawings ® Logabirum in northwest Germany. were handled with Adobe Illustrator CS2. Author's personal copy 272 D. Wulfken,