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Zygospores of selected Trichomycetes in larval Diptera of the families Chironomidae and Simuliidae

     Department of Entomology, Box 340365, Clemson University, Clemson, South Carolina, 29634-0365

	ABSTRACT

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The midgut-inhabiting fungi (Harpellaceae) Harpella melusinae and Stachylina pedifer were induced to form zygospores, using an application of a pH 10 potassium hydroxide solution with culture media. The previously unknown zygospores of S. pedifer are borne perpendicular to the zygosporophore, as in Harpella melusinae. The zygospores of the hindgut-inhabiting species Smittium coloradense, borne obliquely to the zygosporophore (in vivo), are described for the first time.

Key words: aquatic insects, black flies, chironomids, Diptera, fungi, Harpella, Simulium, Smittium, Stachylina, symbionts

	INTRODUCTION

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Fungi of the order Harpellales frequently are found in larval Diptera. These fungi reproduce asexually by trichospores and sexually by zygospores. Trichospores are common and probably constitute the principal means of dispersal and reproduction. Zygospores are less common than trichospores but are found in the branched, hindgut-inhabiting genera, such as Genistellospora, Pennella, and Smittium (Legeriomycetaceae). Rarely are zygospores encountered in the unbranched, midgut-inhabiting genera Harpella and Stachylina (Harpellaceae). Zygospores in aquatic Diptera typically are biconical with thickened walls. Moss et al (1975) described four types of zygospores from Harpellales, based on their position in relation to the zygosporophore. Type I zygospores are borne perpendicular to the zygosporophore and have a median attachment. Type II zygospores are oblique to the zygosporophore and attachment is submedian. Type III zygospores are parallel to the zygosporophore and attachment is median. Type IV zygospores are terminal, with a polar attachment. The role of zygospores in the life history of trichomycetes is not known, although Lichtwardt (1986) hypothesized that the elongate biconical shape is an adaptation for rapid germination during the short transit time through the host gut.

Conjugating thalli sometimes are common locally and temporally in host populations containing midgut-inhabiting Harpellales, but zygospores rarely are found (Lichtwardt 1986). Previously, zygospore formation in the midgut-inhabiting trichomycetes (Harpella melusinae and Stachylina reflexa) was induced by placing conjugating thalli on a microscope slide with distilled water and holding in a moist chamber (Lichtwardt 1986, Lichtwardt and Williams 1988a). However, distilled water does not provide the high pH (9–11) of the larval midgut of certain Diptera (Lacey and Federici 1979). In this paper, we describe a novel method for inducing zygospore production in midgut-inhabiting Harpellales when conjugating thalli are collected. We also provide the first description of zygospores for a typical species, S. pedifer, of the genus Stachylina. Zygospores of Smittium coloradense are described for the first time, and the geographic range of this species is extended.

	MATERIALS AND METHODS

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Larval chironomids (Cricotopus bicinctus or Orthocladius (Eudactylocladius) sp.) containing Smittium coloradense (1 and 28 Apr 1998) and Stachylina pedifer (15 and 28 Apr 1998) were collected from seepage on a bedrock slope at Coneross County Park, Oconee County, South Carolina (N34°38.12', W82°58.20'). Larval black flies (Simulium vittatum cytospecies IIIL-1) with Harpella melusinae were collected on 5 May 1997 from the outlet of Willard's Pond, Clemson, Pickens County, South Carolina (N34°39.40', W82°49.18'). Willard's Pond outlet receives the runoff from a dairy farm. Voucher specimens of the chironomids and slides of the trichomycete zygospores are deposited in the Clemson University Arthropod Collection.

Petri dishes of media were prepared with 3.7 g/L Brain Heart Infusion (BHI) (Difco 0037-15-0) and 15 g/L Bacto agar. The liquid used to rehydrate the medium for H. melusinae and S. pedifer was a pH 10 solution of potassium hydroxide (KOH BHI) made by titrating 10% KOH into water (final ca 0.1 mM). The medium was autoclaved at 121 C for 15 min and aseptically poured into Petri dishes. The pH of the sterile medium was not checked after preparation, but in other preparations of the same kind of medium, the pH was 8.0–8.5 after autoclaving.

The peritrophic matrix or hindgut of each larva was obtained by removing the head and abdominal apex with a scalpel and pulling out the gut with fine forceps. Debris was removed from the peritrophic matrix by pushing it out with a probe or by lifting the matrix in water. Conjugating thalli (found by microscopic examination) of H. melusinae or S. pedifer were placed on the KOH BHI agar and observed with a compound microscope for zygospore formation. Zygospores formed within 48 h on cells with visually homogeneous cytoplasm. Sanitized forceps were used under a dissection microscope to orient and spread the peritrophic matrices on the agar. Cells in which the cytoplasm became nonhomogenous never developed further and were assumed to be dead. An overlay of antibiotics (2000 units Penicillin G and 8000 units Streptomycin HCl/mL) was added to the dishes when bacterial growth became evident. Drops of filter-sterilized pH 10 KOH solution were added as needed to maintain a thin overlay of liquid on the specimens. For photomicrographs, peritrophic matrices were removed from the agar with forceps and placed in aged tapwater on a glass microscope slide. Chironomid hindguts with thalli of Smittium coloradense were squashed to expose the fungi. Semipermanent mounts were made by infusing lactophenol/cotton blue under the coverslips and sealing the edges with nail polish.

	RESULTS

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Zygospores of H. melusinae were Type I, as previously reported (e.g., Lichtwardt 1986). In four out of 10 peritrophic matrices placed on pH 10 KOH BHI, conjugating thalli of H. melusinae formed zygospores. Ten peritrophic matrices with conjugating thalli of S. pedifer were placed on pH 10 KOH BHI, and zygospores were produced on five of these. In two of these matrices, conjugating thalli already had produced trichospores in nonconjugating cells. Stachylina pedifer formed Type I zygospores 48–69 µm x 11–19 µm (n = 10) with a short zygosporophore ca 1–2 µm x 5 µm (n = 3) (Fig. 1). The zygospores we measured might not reflect the final size of the spores because none of the zygospores abscised and, therefore, might not have been mature.

View larger version (127K): [in this window] [in a new window]    FIGS. 1–4. Trichomycetes from larval chironomids (Cricotopus bicinctus or Orthocladius (Eudactylocladius) sp.), Coneross County Park, Oconee County, South Carolina, USA 1. Zygospores of Stachylina pedifer in extracted peritrophic matrix. 2. Zygospore of Smittium coloradense from hindgut. 3. Lateral projections on basal cell of Smittium coloradense. 4. Subapical trichospores of Smittium coloradense. ct = conjugating thalli. Scale bar: 20 µm

Zygospores (in vivo) of S. coloradense (Fig. 2) in our samples were Type II (68–85 µm x 11–17 µm, n = 16), with a narrowed collar (15 x 5 µm, n = 3). The trichospores (n = 13) of S. coloradense were 17–32 µm x 7–15 µm with a 7–13 µm x 2–4 µm collar. Williams and Lichtwardt (1987) gave the collar size as 10–15 µm x ca 3 µm and the trichospore sizes as 17–32 µm x 7–9.5 µm. Other characters of the species that were confirmed by our samples included the small lateral projections on the basal cell (Fig. 3) and subapical trichospores (Fig. 4). Attempts to culture S. coloradense from these samples were unsuccessful. Although the collar lengths on our trichospores were not as long as those described by Williams and Lichtwardt (1987), the combination of morphological characters does not fit other species.

	DISCUSSION

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Zygospores from the genera Harpella and Stachylina are Type I. Zygospores of Harpella melusinae previously had been seen in only seven samples (Lichtwardt 1967, 1986, Moss and Lichtwardt 1977), and all had Type I morphology. Zygospores (Type I) have been reported in only one collection of S. reflexa (Lichtwardt and Williams 1988a); however, S. reflexa is atypical of the morphology of the genus Stachylina (Lichtwardt and Williams 1988a). Stachylina species, such as S. pedifer, normally produce trichospores that detach from the generative cells and have appendages, whereas S. reflexa produces trichospores that disperse while still attached to the disarticulated generative cells and have no appendage. Zygospores of other Stachylina species are unknown. We agree with Lichtwardt and Williams (1988a) that other Stachylina species probably have Type I zygospores.

Our attempts (n = ca 15) to produce zygospores on conjugating thalli of S. pedifer and H. melusinae, using the distilled water method of Lichtwardt (1986), were unsuccessful. Trials with broth of BHI or water agar and pH 10 KOH did not induce zygospore formation. The use of the KOH BHI solution was the only method that promoted the production of zygospores from conjugating thalli of midgut Harpellales. The increased pH or the potassium ion concentration, or both, might be contributing factors. The influence of elevated pH and potassium has been documented for trichospores. Horn (1989) found that potassium and increased pH (followed by reduced pH and potassium) stimulated in vitro germination of trichospores of Smittium culisetae and Smittium culicis.

The genus Smittium has Type II zygospores (Lichtwardt 1986), which, based on published descriptions, have been found in 17 of 55 described species. Smittium coloradense previously has been known from only two locations in Colorado, USA (Williams and Lichtwardt, 1987 Lichtwardt and Williams, 1988b). No zygospores were found in those collections. The present report not only describes the first zygospores of S. coloradense, but also extends the geographic range of the species to the eastern United States (South Carolina).

Zygospores of hindgut-inhabiting Legeriomycetaceae have not been produced in cultured material and still must be obtained from in vivo material. We would not expect the high-pH method (described earlier) to be useful for producing zygospores in hindgut trichomycetes because the hindgut pH is near neutral (Horn 1989, Undeen 1979), although we have not tested high pH or high potassium on these fungi.

Application of the newly described method for obtaining zygospores should yield additional taxonomic information for other midgut-inhabiting Harpellaceae. However, the availability of conjugating thalli is sporadic and limits our ability to clarify the processes of zygospore formation. Further work should clarify the role of the zygospore in the life history and host colonization by trichomycetes. Future work also might enable us to induce conjugating thalli in cultured material so that development of zygospores can be studied in more detail.

	ACKNOWLEDGMENTS

 
This work was supported in part by Grant No. DEB-9629456 from the National Science Foundation to PHA. We thank L. C. Ferrington Jr. and C. N. Watson Jr. for identifying the field-collected chironomids. We thank R. G. Bellinger, M. J. Cafaro, G. R. Carner, R. W. Lichtwardt and M. M. White for reviewing an earlier version of the manuscript. This is Technical Contribution No. 4740 of the South Carolina Agricultural and Forestry Research System, Clemson University.

	FOOTNOTES

 
¹ Corresponding author, cbrd{at}clemson.edu

Accepted for publication August 28, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Horn BW., 1989 Requirement for potassium and pH shift in host-mediated sporangiospore extrusion from trichospores of Smittium culisetae and other Smittium species. Mycological Research 93:303-313

Lacey LA, Federici BA., 1979 Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae). Journal of Invertebrate Pathology 33:171-182

Lichtwardt RW., 1967 Zygospores and spore appendages of Harpella (Trichomycetes) from larvae of Simuliidae. Mycologia 59:482-491

——— 1986 The Trichomycetes, fungal associates of arthropods. New York: Springer-Verlag. 343 p

——— Williams MC., 1983 Two unusual Trichomycetes in an aquatic midge larva. Mycologia 75:728-734

———, ———. 1988a Discovery of sexual reproduction in an unusual new species of Stachylina (Trichomycetes). Mycologia 80:400-405

———, ———. 1988b Distribution and species diversity of trichomycete gut fungi in aquatic insect larvae in two Rocky Mountain streams. Canadian Journal of Botany 66:1259-1263

Moss ST, Lichtwardt RW, Manier J-F., 1975 Zygopolaris, a new genus of Trichomycetes producing zygospores with polar attachment. Mycologia 67:120-127

———, ———. 1977 Zygospores of the Harpellales: an ultrastructural study. Canadian Journal of Botany 55:3099-3110

Undeen AH., 1979 Simuliid larval midgut pH and its implications for control. Mosquito News 39:391-392

Williams MC, Lichtwardt RW., 1987 Three new species of Smittium (Trichomycetes) with notes on range extensions. Mycologia 79:832-838

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