See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/41760759 Spirochete Attachment Ultrastructure: Implications for the Origin and Evolution of Cilia Article in Biological Bulletin · February 2010 DOI: 10.2307/25622856 · Source: PubMed CITATIONS READS 7 95 6 authors, including: M.F. Dolan James Macallister University of Massachusetts Amherst University of Massachusetts Amherst 81 PUBLICATIONS 699 CITATIONS 6 PUBLICATIONS 82 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: A look back at our position paper, "Spirochete round bodies Syphilis, Lyme disease & AIDS: Resurgence of 'the great imitator'?" which is downloaded over 250 times every month from www.environmentalevolution.org View project All content following this page was uploaded by James Macallister on 19 February 2018. The user has requested enhancement of the downloaded file. Reference: Biol. Bull. 218: 25–35. (February 2010) © 2010 Marine Biological Laboratory Spirochete Attachment Ultrastructure: Implications for the Origin and Evolution of Cilia ANDREW M. WIER1, LUCIANO SACCHI2, MICHAEL F. DOLAN3, CLAUDIO BANDI4, JAMES MACALLISTER3, AND LYNN MARGULIS3* 1Pace University, Biology and Health Sciences, 861 Bedford Rd, Pleasantville, New York 10570-2799; 2Electron Microscope Laboratory, Dipartimento di Biologia Animale, Universita` degli Studi di Pavia,University of Pavia, Pavia, Italy; 3Department of Geosciences, University of Massachusetts, Amherst, Massachusetts 01003; and 4DIPAV, Sezione di Patologia Generale e Parassitologia, Universita` degli Studi di Milano, Milan, Italy Abstract. The fine structure of spirochete attachments to Introduction the plasma membrane of anaerobic protists displays varia- tions here interpreted as legacies of an evolutionary se- Spirochetes, a cohesive phylum of gram-negative eubac- quence analogous to that from free-living spirochetes to terial organo-chemoheterotrophs (Margulis and Chapman, undulipodia (eukaryotic “flagella” and homologous struc- 2010) tend to swim in synchrony and attach to each other tures). Attached spirochetes form a vestment, a wriggling and to other live cells. They penetrate high-viscosity gels, fringe of motile cells at the edge of the plasma membrane of muds, and tissues. Spirochete attachment to the membranes unidentified cellulolytic protist cells in the hypertrophied of cellulolytic termite hindgut protists takes many forms— hindgut of the digestive system of Mastotermes darwinien- from loose and casual to complex, repeated and tight, per- sis, the large wood-feeding termite from northern Australia. manently ectosymbiotic structures. Heterologous spiro- chete-membrane attachments to eukaryotic cells in an From the membrane extend both undulipodia and a complex anoxic, microoxic-to-oxic gradient environment are de- of comparably sized (10–12 ␮m ϫ 0.2–0.3 ␮m) ectosym- scribed here. The termite hindgut is anoxic only in its biotic spirochetes that resembles unruly ciliated epithelium. center, while the edges of the gut are microoxic (Brune et In the intestines are helical (swimming) and round-body al., 1995). Our goal here is to interpret structural variations morphotypes. Round bodies (RBs) are slow or immotile in both the spirochetes and the cells to which they attach in spirochetes, propagules known to revert to typical swim- a specific evolutionary context. ming helices under culture conditions favorable for growth. In study of hindgut microbes of the unique wood-feeding The surfaces of both the spirochete gram-negative eubacte- termite Mastotermes darwiniensis (Froggatt), the only ex- ria and the parabasalid protists display distinctive attach- tant member of its genus, we sought ecto-and endosymbi- ment structures. The attached hypertrophied structures, onts in and on one of its protists: the giant trichomonad some of which resemble ciliate kinetids, are found consis- Mixotricha paradoxa (Sutherland). M. paradoxa, which in- tently at sites where the spirochete termini contact the gests pieces of wood through its posterior although it swims protist plasma membranes. with its anterior forward, comprises a motility symbiosis with at least three different types of spirochetes and their attachment structures (Cleveland and Grimstone, 1964; Wier et al., 2001; Wenzel et al., 2003). These spirochetes Received 14 September 2009; accepted 16 November 2009. form a fringe over the entire surface of the cell. The coor- * To whom correspondence should be addressed. E-mail: dinated beating of this fringe of motile cells propels M. [email protected] Abbreviations: RBs ϭ spirochete round bodies, cysts, L-forms, prop- paradoxa through the liquid that fills the termite’s gut. The agules, resting stages, vesicles, granules, etc. See Margulis et al. (2009) for spirochetes include a thin treponeme morphologically indis- explanation. tinguishable from Treponema pallidum,aBorrelia-like me- 25 26 A. M. WIER ET AL. dium-sized 5:4:5 or 4:8:4 spirochete attached at the poste- mite guts were dissected and their contents harvested from rior periphery of the cell’s wood ingestive zone, and live specimens of M. darwiniensis workers (pseudergates). Canaleparolina sp., a larger and longer spirochete (26–28 The head of the termite was held in blunt forceps as fine ␮m length ϫ 0.4–0.7 ␮m width) known only from two forceps were used to extract whole intestines that were remotely separated localities (Darwin, Australia, and St. immediately prefixed in Karnovsky’s fixative in cacodylate John, US Virgin Islands, in the Caribbean; Wier et al., buffer (pH 7.2). After postfixation in 2% OsO4 for 1.5 h, 2001). These species designations are based on transmission samples were washed in cacodylate buffer, dehydrated electron microscopic cross-sections of the spirochetes, not through an ethanol series, transferred to propylene oxide, on gene-sequence-based identification, although our results and embedded in Epon 812. The embedded samples sent to are consistent with other studies of this microbial symbiosis the first author (A.M.W.), University of Wisconsin, Mad- (Wenzel et al., 2003; Brugerolle, 2004). Our plan was to ison,Wisconsin, had been fixed in 2.5% glutaraldehyde in study, by morphological correlation at the transmission phosphate buffered saline. Within 4 weeks after fixation electron microscopic level, endo- or ectonuclear bacterial they were postfixed in osmium and embedded in Epon resin symbionts and/or contractile karyomastigont that we saw in polymerized for 48 h at 60 °C. Three blocks were retained living M. paradoxa cells. The karyomastigont, an organellar by L.S. (Pavia) and two mailed to A.M.W. system that includes the nucleus, the nuclear connector, and Thin sections, cut with a diamond knife on a Reichert- kinetosome-centrioles in many protoctist and animal cells, Jung ultracut microtome, were stained with saturated uranyl is a conspicuous component of the cytoskeleton. While acetate followed by Reynolds lead citrate and examined neither bacterial nuclear symbionts (Dolan et al., 2004) nor with a Zeiss 900 (at 80 kV) or a Siemens AMW2 (at 30 kV) the morphological basis of the contractile portion of the electron microscope. nuclear connector/“rhizoplast” (of the karyomastigont) was imaged, we discovered a plethora of different spirochetes in Results contact with the outer membranes of protists. We inter- preted these images as putative stages of increasing inti- The preservation and fine structure resolution of the tis- macy of the helical, motile eubacteria attached to amito- sue are excellent. However, in most cases the genus and chondriate parabasalids. We describe and interpret here species of protists to which the spirochetes are attached are fewer than a dozen from a larger set of micrographs in an not known. Nor have the bacterial species been identified attempt to reconstruct a plausible evolutionary sequence. below the level of phylum. The eukaryotic microbes, ami- tochondriate heterotrophs, are wood-ingesting motile pro- Materials and Methods tists (phylum Archaeprotista, class Parabasalia, kingdom Protoctista), whereas the attached prokaryotes are recog- Mastotermes darwiniensis is the remarkable sole survivor nized by morphology and motility (live, videography, light of Paleocene termites of the family Mastotermidae. Masto- and electron microscopy) as spirochetes: phylum Spirochae- termitids, like their wood-feeding cockroach blatterid an- tae of the kingdom Prokaryotae (Margulis, 2000; Margulis cestors, lay eggs in packaged masses rather than singly; and Chapman, 2010). The cellulolytic protists and their therefore zoologists consider this family of “lower termites” attached motile eubacteria here share the same habitat— the to be the earliest evolved in the Isoptera lineage. Individual intestine of the Australian dry-wood-ingesting termite Masto- insects, members of the mastotermitid genus, are well pre- termes darwiniensis. The observations of live organisms are served as abundant fossils, especially in amber. They are confirmed by ultrastructure. The eubacterial-parabasalid known worldwide—for example, Mastotermes mexicanus physical associations must be permanent, or at least of long and M. dominicus from the American tropics. The relevance duration, because they have been consistently observed of the availability of limited live mastotermitids with an since 16-mm black-and-white cine´ films were made of M. immense fossil record was brought to our attention by D. darwiniensis microbes by Harvard