INTERNATIONALJOURNAL OF SYSTEMAT~CBACTERIOLOGY, July 1988, p. 291-302 Vol. 38, No. 3 0020-7713/88/030291-12$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Morphology as a Basis for Taxonomy of Large Spirochetes Symbiotic in Wood-Eating Cockroaches and Termites: Pillotina gen. nov., norn. rev.; Pillotina calotermitidis sp. nov. norn. rev.; Diplocalyx gen. nov. nom. rev. ; Diplocalyx calotermitidis sp. nov. , nom. rev.; Hollandina gen. nov., nom. rev.; Hollandina pterotermitidis sp. nov. , norn. rev. ; and Clevelandina reticulitermitidis gen. nov. sp. nov. DAVID BERMUDES,’? DAVID CHASE,2$ AND LYNN MARGULIS1* Department of Biology, Boston University, Boston, Massachusetts 022 15,’ and Division of Surgical Oncology, Department of Surgety, School of Medicine, University of California Davis Medical Center, Davis, California 9561 1‘ The purposes of this paper are (i) to present a framework for the morphometric analysis of large uncultivable spirochetes that are symbiotic in wood-eating cockroaches and termites; (ii) to revive, in accordance with the rules of the International Code of Nomenclature of Bacteria, the names of three genera (Pillotina, Diplocalyx, and Hollandina) and three species (Pillotina calotermitidis, Diplocalyx calotermitidis, and Hollandina pteroter- mitidis) for the same organisms to which the names were originally applied, because these names were not included on the 1980 Approved Lists of Bacterial Names; and (iii) to formally propose the name Clevelandina reticulitermitidis for a new genus and species of spirochetes from the termite Reticulitermes tibialis. None of these genera and species has been cultivated either axenically or in mixed culture; hence, all are based on type-descriptive material. Large spirochetes living under the anaerobic or microaero- uncultivable spirochetes that are symbiotic in wood-eating philic conditions in intestines of wood-eating cockroaches termites and cockroaches. and termites have never been cultivated (31). A new genus In this paper we revive the genera “Pillotina,” “Diplo- and species, “Pillotina calotermitidis,” was proposed in calyx,” and “Hollandina” and the species “Pillotina calo- 1967 by Hollande and Gharagozlou (10) for a spirochete from termitidis ,” ‘ ‘Diplocalyx calotermitidis,” and “Hollandina the termite Calotermes praecox. Another new genus and pterotermitidis,” in accordance with the rules of the Inter- species, “Diplocalyx calotermitidis,” was proposed for a national Code of Nomenclature of Bacteria (24), for the separate morphotype from the hindgut of the termite Cab- same organisms to which the names were originally applied, termesfiavicollis by Gharagozlou (8) in 1968. A third genus because they were not included on the 1980 Approved Lists and species, “Hollandina pterotermitidis” from the hindgut of Bacterial Names (30). We fornially propose the name of the termite Pterotermes occidentis, was described by To bbClevelandinareticulitermitidis” for the type species of the et al. in 1978 (31). The genus “Clevelandina” has been new spirochete genus “Clevelandina” from the termite informally proposed previously (26). Reports on these types Reticulitermes tibialis. of spirochetes have been reviewed by Breznak (6). The work of Hollande and Gharagozlou is of particular MATERIALS AND METHODS interest because hollow tubules, 25 nm in diameter, were Sources of termite spirochetes. Termites were collected as reported to be in the protoplasmic cylinder. Immunocyto- indicated in Table 1. They were maintained in a laboratory, chemical investigations demonstrate the presence of antitu- and the guts were removed for light and electron micro- bulin immunoreactivity in these large spirochetes (25). Fur- scopic observations (31, 33). ther investigations of small cultivable spirochetes have Light microscopy. For observation of living material, resulted in the isolation of tubulinlike proteins (1).However, freshly dissected guts were placed upon a glass microscope not all large spirochete morphotypes appear to have cyto- slide, covered with 1 or 2 drops of Trager solution A or plasmic tubules. Characterization of spirochetes with or Trager solution U (34), and pierced with a dissection needle. without tubules requires distinctions among morphotypes. A cover glass was then sealed over the preparation with We have examined the following four species of subterra- petroleum jelly. nean and dry-wood-eating termites by electron microscopy: Electron microscopy. For preparation of thin sections, Pterutermes occidentis, Reticulitermes hesperus, Reticuli- entire termite guts were fixed with 3% glutaraldehyde in termes tibiafis, and Incisitermes minor. Our observations 0.025 M phosphate buffer at pH 6.8, postfixed with 1% provide a framework for the morphometric analysis of osmium tetroxide in the same buffer, dehydrated in acetone, and embedded in Spurr resin by using the methods of Bloodgood (3) and Bloodgood and Fitzharris (4) as modified * Corresponding author. by To et al. (31) (2 to 5% tannic acid was added to the fixative t Present address: Center for Great Lake Studies, University of for some of the samples and the impregnation time was Wisconsin-Milwaukee, Milwaukee, WI 53204. extended to 24 h). Thin sections were stained first with 2% f Deceased. uranyl acetate and then with 2% lead citrate. 291 292 BERMUDES ET AL. INT. J. SYST.BACTER~OL. TABLE 1. Sources and identification of North American termites used in this study Isopteran insect Locality Collected by: Identified by: Subterranean termi tes (Reticuli termi tidae) Reticulitermes hesperus San Diego, Calif. L. Margulis D. Chase Reticulitermes tibialis Joshua Tree National Monument, Nev. D. Chase D. Chase Dry-wood-eating termites (Kalotermitidae) Pterotermes occidentis Tucson, Ariz. L. Margulis W. Nutting Incisitermes (Kalotermes) minor Los Angeles, Calif. D. Chase D. Chase Incisitermes (Kalotermes) snyderi Naples, Fla. S. F. Bermudes P. Luykx Morphometrics. A total of 15 morphological criteria were diameter (criterion 8) is illustrated in Fig. 1C. By using a measured (Table 2 and Fig. 1). The averages reported below ratio, comparisons could be made among spirochetes of represent measurments of all material from the original different diameters. Criterion 9, the angle subtended by the publications and from 3 to 20 examples of each morphotypes flagella, is a measurement of the distribution of the flagella in from the termites which we examined. Diameters (criterion the periplasm (Fig. 1D). Flagellar bundles (criterion 10) were 1) in oblong cells were measured at the narrowest and widest deemed to be present if the angle subtended by the flagella points enclosed by the cell membrane. In crenulated cells, was less than 180" and in areas where the flagella were not measurements were made at the tips of the crenulations. The present, the outer membrane was in close apposition to the number of flagella (criterion 2) is reported as the number inner membrane (Fig. 1E). In such cases, a concavity was inserted at each end, approximated by the median number formed by the outer membrane near the junction between observed. The number of flagella observed could range from the flagellar bundle and the protoplasmic cylinder. An angle zero (beyond the most distal insertion) to twice the number was formed by three points. Two points were formed by the of flagella inserted at one end, where, toward the center, intersection of a straight line with the outermost region of the flagella overlapped with the flagella from the other end. outer membrane (Fig. lE, line A, points 1 and 2). A third Previous descriptions (8, 10, 26, 31) have reported the point was formed by the intersection of a line perpendicular maximum number observed in cross sections. The sillon (10) to line A (line B) with the outer membrane at a distance or groove (criterion 3) is an invagination of the outer maximal from line A. The angle of concavity was measured membrane toward the inner membrane (Fig. 1B). This struc- as the angle formed by the intersection of a line between ture provides a reference point for the description of other points 1 and 3 (line C) and a line between points 2 and 3 (line structures. Crenulations (criterion 4) are conspicuous folds D). The angle was less than 180" when a bundle was present. in the outer membrane (Fig. 1B). Criteria 5 through 7 deal Cytoplasmic tubules (criterion 14) were small, hollow, with the presence of coatings on the inner and outer mem- walled structures as observed by electron microscopy. The branes of the gram-negative cell walls (i.e., the glycocalyx or polar organelle (synonym, polar membrane) (criterion 15) sheath); the use of a ratio results in partial compensation for was an electron-dense lamina located on the inside of the slightly oblique sections. When such surface layers are inner membrane toward the distal end of the cell (Fig. 1B). absent, the ratio of their thickness to the thickness of the membrane itself is zero. Absolute values are also given RESULTS AND DISCUSSION below. Spirochetes and protoplasmic cylinder cross sections of oblong shape were measured at both their widest (Fig. 1C) Morphological characteristics. The major criteria (criteria 1 and narrowest points. The method for determining the ratio through 10) for distinguishing spirochete morphotypes are of the protoplasmic cylinder diameter to the overall cell listed in Table 2. Criteria 11 through 15 provide less impor- TABLE 2. Morphological criteria for assigning spirochetes to genera Criterion Figure Overall range in illustrating