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The status and characterization of Enteroramus dimorphus: a xylose-fermenting yeast attached to the gut of beetles

     Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

Merlin M. White

     Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045

Nhu H. Nguyen
Meredith Blackwell ¹

     Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

	ABSTRACT

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Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.

Key words: Ascobotryozyma americana, Ascobotryozyma cognata, Botryozyma nematodophila, evolution, LSU rDNA, ITS, Passalidae, symbiosis, yeast

	INTRODUCTION

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Passalid beetles (Passalidae: Odontotaenius disjunctus) are known to harbor three species of Eccrinales (Trichomycetes) and a fourth organism, a yeast-like fungus previously regarded as of unknown affiliation. The organisms are partitioned within specific regions of the long recurved beetle gut (Lichtwardt et al 1999). The mechanism by which the organisms are partitioned in the gut is unknown, but the gut regions clearly are differentiated morphologically and, probably, physiologically. The three eccrinaleans include Leidyomyces attenuatus located at the anterior-most region of the hindgut or the ileum, often occurring either in whorled clusters of thalli with a shared common holdfast structure or as individual attenuated thalli in the posterior ileum; an undescribed taxon, "Heymons’ eccrinid," scattered throughout the smoother hindgut region posterior to the ileum; Passalomyces compressus (Trichomycetes: Eccrinales), posterior-most and attached on the gut lining or anal plates (See FIG. 17 in Lichtwardt et al 1999). Enteroramus dimorphus, the non-trichomycete, yeast-like fungus, occurs in the same gut region as the undescribed taxon, attached by a holdfast at the "suture" lines that are typical of the chitinous lining of this section of the hindgut.

In another ongoing study designed to discover yeasts in the gut of a variety of beetle species, a yeast closely related to Pichia stipitis was isolated consistently from the passalid beetle, O. disjunctus. Isolation of the yeast from passalids in the eastern United States (Pennsylvania, South Carolina, Georgia, Louisiana) suggests that they are associated with the beetle throughout much of its broad range in eastern North America (Suh et al 2003). Phylogenetic analyses using SSU and LSU ribosomal DNA of (i) Pichia stipitis, (ii) the yeast isolates from eastern United States passalids and (iii) the Kansas isolate of Enteroramus dimorphus, indicated that they all are very closely related if not conspecific.

Members of the P. stipitis clade are notable among yeasts for their ability to ferment and assimilate xylose (Kurtzman 1990, Jeffries and Kurtzman 1994). Residues of the five-carbon sugar xylose form the backbone of a major plant cell-wall component, hemicellulose, and P. stipitis has attracted wide attention in efforts to improve fermentation of plant residues for use as fuel alcohol (van Dijken et al 1986, Jeffries and Jin 2000, Ward and Singh 2002). It is assumed that fermentation of xylose would benefit wood-ingesting beetles (Suh et al 2003).

In this report we compare isolates of P. stipitis to the yeasts from passalid beetles, present an emended description of E. dimorphus and discuss its nomenclature. The symbiotic phase of a passalid isolate is compared to yeasts associated with nematodes.

	MATERIALS AND METHODS

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Host beetles, yeast isolation and culture. – A culture (KS-42-W2) established from the holotype material of Enteroramus dimorphus from Kansas (Lichtwardt et al 1999) was compared with other yeasts isolated from O. disjunctus in Georgia, South Carolina, Louisiana and Pennsylvania (TABLE I). Vouchers of the Kansas insects and slides of their trichomycetes and yeasts are in the collection of MMW. The yeast culture (KS-42-W2) originally deposited at the University of Kansas Culture Collection has been accessioned into the Agricultural Research Service (ARS) Culture Collection (NRRL Y-27535). Isotypes of E. dimorphus are at FH (Lichtwardt et al 1999). Cultures of the passalid yeasts from the eastern United States also were deposited in the ARS Culture Collection (NRRL Y-27547–NRRL Y-27555). Beetles were dissected as described previously, and gut segments were crushed in a saline solution with a pipette tip (Lichtwardt et al 1999, 2001, Suh et al 2003). The saline solution containing inoculum was streaked onto the surface of an acidified YM agar plate (Difco YM broth, 2% plain agar, adjusted to pH 3.5 with HCl) and incubated at 25 C. Single colonies were streaked for purification at least two times, and the purified cultures were maintained on ME agar (Difco malt extract, 2% plain agar) (Suh et al 2003). Morphological observations and metabolic tests were performed on the yeasts by the methods described by Kurtzman and Fell (1998) and Barnett and colleagues (2000). Observations on Pichia stipitis were reported previously (Kurtzman and Fell 1998, Barnett et al 2000).

	RESULTS

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Yeast cultures. – Yeasts isolated from the gut and external surfaces of about 22 adult passalid beetles (O. disjunctus) were established in pure culture on YM agar. A single yeast was isolated from a beetle, and only one beetle failed to yield a yeast culture. The Kansas isolate (E. dimorphus) was designated PASS5 based on its unique LSU rDNA D1/D2 loop sequence that was 1 bp different from the other passalid isolates (PASS1); the SSU rDNA sequence was identical to the PASS1 isolates and 1 bp different from P. stipitis. The yeast isolates from all other passalid beetles that were collected in Pennsylvania, South Carolina, Georgia and Louisiana had identical sequences when the D1/D2 loop of the LSU rDNA, SSU rDNA, and ITS were compared (Suh et al 2003). There were few morphological differences among the passalid isolates and P. stipitis (TABLE II). Physiological differences among the passalid isolates and P. stipitis included differences in six of the carbon and one of the nitrogen assimilation tests (Barnett et al 2000, Suh et al 2003).

Morphology in symbiotic association with Odontotaenius disjunctus. – FIGS. 1, 2. Septate filaments deeply branched, up to 410 µm in length. The filaments of the thalli forming tufts that attach along furrows of the smooth hindgut lining. The columnar bases (15–21 x 8–10 µm) of the filaments are the attachment sites. The yeast stage initiates from ovoid cells (2.5–3 x 4.5–6 µm) that develop by budding from the filaments in moist chamber or on agar media.

View larger version (107K): [in this window] [in a new window]   FIGS. 1, 2. Enteroramus dimorphus in symbiotic association with Odontotaenius disjunctus. 1. Holdfast cells (arrow) at the base of two attached filamentous thalli. Scale bar = 10 µm. 2. Filamentous thalli from water mount showing budding cells. Scale bar = 20 µm. (Photomicrographs by MMW, previously published Mycologia 91:698, used with permission of MMW).

View larger version (61K): [in this window] [in a new window]   FIGS. 3, 4. Yeast culture of E. dimorphus on cornmeal agar at 25 C after 7 d. 3. Budding cells. 4. Ascus with two hat-shaped ascospores (arrow). Scale bars = 5 µm.

Closest known taxa and base pair comparison of genes with E. dimorphus. – Pichia stipitis (LSU rDNA [U45741], 1n [unreadable base] bp; ITS [AB054115], 7 bp; Kurtzman and Robnett 1997), PASS1 (LSU rDNA [AY227720], 1 bp; ITS [AY227900], 1bp; Suh et al 2003)

DNA sequences. – Sequences deposited in GenBank include SSU rDNA (AY227899), LSU rDNA (AY227722), and ITS including 5.8S rDNA (AY227902).

	DISCUSSION

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Evidence from DNA, morphology and physiology indicates that all yeasts discussed in this report are related closely. While it might be assumed that all the isolates could be included as P. stipitis, we have not made a new combination for E. dimorphus or even included it in the synonymy of P. stipitis for two reasons: First, some strains of passalid yeasts might have become isolated within the host from other saprobic yeast populations; second, a revision of yeast nomenclature is needed to establish monophyletic taxa. The genus Pichia E.C. Hansen with P. membranifaciens as the type species is a prime candidate for taxonomic treatment, and many distinct clades need attention. Pichia membranifaciens is not a member of the P. stipitis clade; therefore a simple transfer of E. dimorphus to Pichia is not an option. Enteroramus dimorphus should be placed among its close genetic relatives in a clade containing P. stipitis and the other xylose-fermenting members of that clade.

In addition to the one base pair differences in LSU rDNA observed among P. stipitis, PASS5 and PASS1 isolates (TABLE I), small morphological (TABLE II) and physiological differences are present. Fermentation profiles for the three groups (P. stipitis, PASS5, PASS1) were identical. Carbon and nitrogen assimilation, vitamin production and growth temperature differences were trivial, usually only by degree and rate of the reactions. In total there were only seven differences in carbon and nitrogen assimilation tests when the reactions are considered positive (including delayed, weak) or negative. P. stipitis strains (Barnett et al 2000) and PASS1 (Suh et al 2003) were more similar in their carbon assimilation profiles, but only PASS1 and PASS5 synthesized biotin weakly. By comparison, the data reported for multiple isolates of P. stipitis (Kurtzman and Fell 1998) indicated variable reactions for 11 carbon assimilation tests when the reactions are considered as positive or negative. Variation in assimilation capacity among the groups, therefore, is less than it is for multiple isolates of P. stipitis, that are equivalent to the strain differences commonly observed within species.

Yeasts seldom have been observed in situ in their natural habitats because they are microscopic and usually observed in culture. The holdfast of the passalid beetle-associated thalli of E. dimorphus might be unusual (FIGS. 1, 2, Lichtwardt et al 1999), but a parallel exists between the passalid yeast and an unrelated group of yeasts known from complex associations with nematodes and insects in plants. Botryozyma nematodophila was described in association with nematodes and Drosophila in grapes with the sour-rot disease in Italy (Smith et al 1992); the related teleomorphs, Ascobotryozyma americana and A. cognata, were discovered on the surface of nematodes in the galleries of the poplar borer, Saperda calcarata in Washington and Idaho (Kerrigan et al 2001, 2003). These closely related yeasts have branched thalli of determinate growth, composed of pseudohyphae. The thalli were seen attached to the nematode surfaces by specialized holdfast cells with two perpendicular branches of equal size that clasped the surface of the nematodes. The exact method by which the yeasts were attached to the nematodes is unknown, but nematode cuticles were not penetrated. The nuclei of mature basal cells of A. americana were degenerate, a condition that Kerrigan and her colleagues (2001) interpreted as an indication that the cells were not active in penetration or conduction of nutrients.

When other yeasts are examined in their natural substrates, it is possible that they have more complex morphologies than we now know; additional observations of suspected yeast substrates using both light and electron microscopes might be profitable in determining such conditions (Lichtwardt et al 1999; Kerrigan et al 2001, 2003).

	ACKNOWLEDGMENTS

 
We are grateful to Drs Eddie Beard and Will Reeves, Clemson University, who isolated yeast cultures from South Carolina. Drs Robert Lichtwardt, Matías Cafaro, J.K. Misra, and Hiroki Sato helped to collect passalids near Lawrence, Kansas, and Dr Douglas Tallamy, University of Delaware, provided insect specimens from Pennsylvania. Dr Julia Kerrigan generously gave us access to unpublished results, and Dr Cletus Kurtzman accessioned the yeast isolates into the ARS collection. We appreciate the untiring commitment of undergraduate students Christine Ackerman, Katie Brillhart, Cennet Erbil and Amy Whittington, who helped perform the time-consuming work of isolating, culturing and photographing the yeasts. We acknowledge the use of the Socolofsky Microscopy Center and the expert assistance of Ying Xiao for preparation of the photographic plates. This work was supported by grants from the National Science Foundation (DEB-0072741 with two REU supplements to MB and DEB-9521811 to Robert Lichtwardt and Leonard Ferrington Jr.).

	FOOTNOTES

 
Accepted for publication December 7, 2003.

¹ Corresponding author. E-mail: mblackwell{at}lsu.edu

	LITERATURE CITED

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