J. zyxwvutsrqponmlkjihgfe Euk. Microbrol., zyxwvutsrqponmlkj 45(1J, 1998 zyxwvutsrqponmlk pp. zyxwvutsrqponml 64-70 zyxwvutsrqponm 0 1998 by the Society zyxwvutsrqponmlkj of Protozoologists Epibiotic Bacteria on Several Ciliates from Marine Sediments SLAVA S. EPSTEIN,*,' DENNIS A. BAZYLINSKI,** and WILLIAM H. FOWLE*** *Marine Science Center, Northeastern University, East Point, Nahant, Massachusetts 01 908. USA, and **Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Arnes, Iowa zyxw 5001 I, USA, and ***Electrun Microscopy Center, Department of Biology, Northeastern University, Boston, Massachusetts 02 115, USA ABSTRACT. Bacterial epibionts were observed on the surface of the marine sediment ciliate Geleiu fossata. Rod-shaped bacteria, from 2-10 X lo3 per ciliate, were universally positioned in ciliated grooves, in apparent spatial association with dikinetids. SEM and TEM examination of the ciliates confirmed that a tight affiliation exists between the epibiotic bacteria and ciliate cortex ultrastructures. These observations, as well as the distinct bacterial distribution pattern over ciliate surface, suggest that there is a close epibionthost physiological integration. Epibiotic bacteria were also observed on the surfaces of other sediment ciliates from the genera Loxuphyllum, Tracheloraphis, Geleia, Paraspathidium, and Cyclidium. These findings indicate that the bacteriallprotozoa associations are widespread in the marine benthic environment. The potential benefits for both epibionts and their hosts are discussed. Supplementary key words. Benthos, chemolithotrophs, oxic-anoxic interface, prokaryotes, protozoa, symbiosis. LOSE associations between Protozoa and bacteria have been C recognized for over a century [5, 61 and include examples of parasitism, commensalism, and mutualism. In many of these associations, the protozoan hosts are inhabited by ecto- and/or endobiotic bacteria. The most studied examples are the cyto- plasmic and endonuclear bacteria occumng in Paramecium and other Protozoa (reviewed in [32]). Some cytobionts, termed xe- nosomes [42], are infectious and parasitic. Other associations appear to be more mutualistic in nature, such as those between methanogenic and other anaerobic bacteria and anaerobic cili- ates [24, 471. These symbioses elegantly resolve some of the physiological constraints of the above organisms imparted by the nature of their habitat [25]. In the anaerobic environment, be it marine sediments [26, 451, freshwater chemically-stratified systems [16, 171, the reticulo-rumen of some mammals [29, 471, the hind gut of insects [28, 331, or landfill sites [24], Protozoa- bacteria synergism and symbiosis seem to be the rule rather than exception. In most cases, the close relationship is critical for the energy metabolism for both organisms [25]. Although the most-studied examples of protozoan-bacterial synergisdsymbiosis involve anaerobic microorganisms, there is no a priori reason why functionally important associations between bacteria and Protozoa should not exist in well-oxygen- ated or microaerophilic environments as well. In the latter sit- uation, examples are largely limited to two bacterial-protozoan relationships, both involving epibiotic bacteria, possibly sulfur- oxidizers, and two marine ciliates, Kentrophoros [23] and Zoothamnium [2]. In this paper, we present evidence to support the idea that such associations are common in the marine ben- thic environment. We describe associations between what ap- pear to be ectosymbiotic bacteria and several marine sediment ciliates and provide SEM and TEM ultrastructural evidence for close integration of epibiotic bacteria and their hosts' cortical structures. MATERIALS AND METHODS Source of organisms. Ciliates were obtained from sediment samples taken at various times of the year from a marine sandy tidal flat, near the Marine Science Center, Northeastern Uni- versity, Nahant, MA, 10 km north of Boston. The study site is described elsewhere [14, 151. Identification of ciliates. Sediments were preserved with Bouin's fixative (37% buffered formaldehyde, saturated with picric acid, with glacial acetic acid added to a 1% final con- centration prior to fixation; these and other chemicals were, unless otherwise specified, from Sigma, St. Louis, MO). Cili- ates were extracted by repetitive sample resuspension and de- I To whom correspondence should be addressed. Telephone: 617-581- 7370; Fax: 617-581-6076; Email: zyxwvutsr sepstein@lynx.dac.neu.edu cantation. Extracted ciliates were concentrated on 1- to 2-pm pore size Millipore nitrate cellulose filters and were silver-im- pregnated with Protargol (Roboz Surgical Instrument Co., Rockville, MD) as described in [36]. Microscopic observations were made using Zeiss Standard or Axiophot microscopes. Original species description and recent reviews [7, 11, 19, 30, 37, 40, 411 were consulted for identification and taxonomy. Epifluorescence observations. Sediment samples were pre- served with 2% glutaraldehyde (final concentration in the sed- iment). The ciliates were extracted, purified, stained with DAPI (4',6 diamidino-2-phenylindol) or dual stained with DAPVFITC (fluorescein isothiocyanate), and concentrated on 2-pm pore size black polycarbonate membranes (Polysciences, Warring- ton, PA) after Epstein [13]. Observations and microphotography were carried out using Zeiss Axiophot microscope equipped for epifluorescence (Zeiss filter blocks 9108460194 for DAPI (365/ 400/450 nm excitation filterheam splitterharrier filter, respec- tively) and 9 108460019 for FITC (485/505/>530 nm filters)). SEM. Ciliates were extracted using a sea ice technique [44], collected and transferred to a vial with aid of a micropipette, preserved with EM-grade glutaraldehyde (2% final concentra- tion), concentrated on 2-pm pore size polycarbonate mem- branes, and post-fixed in 1% osmium tetroxide. Following post- fixation, the polycarbonate membranes were dehydrated using a graded series of ethanol and critically point dried from CO?. Membranes were then mounted on a specimen stub with a car- bon adhesive tab, sputter coated with 15 nm gold-palladium and examined using an AMR- 1000 scanning electron microscope. TEM. Sediments were preserved as for epifluorescence ob- servations. Organisms were separated from the sediment as for the identification of ciliates. Target ciliates were collected by a micropipette and transferred to a vial containing 4.0% glutar- aldehyde, 0.3M sucrose, and 0.1M cacodylate buffer (pH 7.4) for 2 h at 4" C. The ciliates were then washed twice with 0.1M cacodylate buffer containing 0.3M sucrose, post fixed for 1 hour in cacodylate buffered 1 % osmium tetroxide, dehydrated through a graded series of ethanol, and infiltrated with increas- ing ratios of Spurrs resin to 100% ethanol. After infiltration in 3 one-hour changes of pure Spurrs resin, individual ciliates were embedded in flat embedding molds to allow for orienta- tion, and polymerized at 60" C for 24 hours. Thin sections cut on a diamond knife were collected, stained with uranyl acetate and lead citrate, and examined in a Zeiss EM-I0 transmission electron microscope. RESULTS Identification of ciliates. The ciliates examined in this study were identified as follows: Loxophyllurn setigerum, Tvachelor- aphis primitarum, Tracheloraphis zyx sp.. Geleia fossata, Geleia 64