Proc. Nail. Acad. Sci. USA Vol. 76, No. 5, Pp. 2299--2302, May 1979 BotanN Tabanomyces milkoi (Dudka and Koval) emended, genus novum, a fungal pathogen of horseflies (/deerflies/biocontrol/loiasis/phylogenetic significance) J. N. COUCH*, R. V. ANDREEVAt, M. LAIRDt:, AND R. A. NOLAN§ *I)epartrment of Botany, Universitv of North Carolina, Chapel Hill, North Carolina 27514; tDepartment of Parasitology, Zoological Inistitote. Aca(leinv of Sciences, Ukrainian SSR, Kiev, U.S.S.R.; Research Unit on Vector Pathology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A IC 5S7; andc §Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada Al13 3X9 Contributed by John N. Couch, March 1, 1979

ABSTRACT A fungus originally described as a new species of Coelomomyces (C. milkoi) from larval Tabanidae in the Ukraine, USSR, is transferred to a new genus, Tabanomyces, and described in detail. The zygospores germinate to form a linear four-celled conidiophore, each cell producing a spherical ejected as in , thus confirming that the organism belongs in the . The resemblance between the conidiophore of Tabanomyces milkoi and the I' l(.. I. la'o i It(I . osIII Iu/i., 1. ed /mI/ I- of the rusts supports the old concept that the rust fungi PILV'ccs 01'1 n SUcaI( represenlts nun. P'holwgrasll hy may be related to the Entomophthorales. HAN. In 1973 Dudka et al. (1) described as Coelomomyces milkoi MATERIALS AND METHODS Dudka and Koval a fungus from the hemocoel of larval of the zygospores ("sporangia") of the fungus from horseflies (Tabanus autumnalis and Chrysops relictus) from larval T. autumnalis was obtained by the same methods used damp soil at the edge of large artificial ponds near Kiev, Uk- by Couch (6) for the germination of the sporangia of Coelo- raine, USSR. In that paper, only the and "sporangia" momyces. A small section of a dried, parasitized Tabanus larva were described. Dubitskii et al. (2) succeeded in germinating packed with the zygospores was placed on a clean slide and, the "sporangia" and reported the production and emergence with the aid of a dissecting microscope, a piece about the size of zoospores. Nam and Dubitskii (3) in 1977 transferred this of a pinhead was cut off. This piece was placed in a small drop fungus to the order Entomophthorales because of the presence of distilled water on a slide, and the zygospores ("sporangia') of septa in the mycelium and the fusion of hyphal bodies to were dispersed in the drop. Some zygospores floated on the form a zygospore-the "" of previous studies. They surface, and others remained at the bottom of the drop. The also proposed a new name, Pseudocoelomomyces milkoi, to slide was incubated in a damp chamber at room temperature replace Coelomomyces milkoi. (20-26°C). In germination studies with various species of A septate mycelium and gametangial copulation to form a Coelomomyces, Couch (6) had found that thick-walled spo- zygote are characteristics of both the Entomophthorales and rangia of certain species, such as Coelomomyces punctatus, the order Lagenidiales. Because the diagnostic characteristic germinated after being in water on a slide for 48 hr, whereas of the Entomophthorales is asexual reproduction by forcibly sporangia from certain other species required as much as 7 days ejected conidia, the ordinal position of "Pseudocoelomomyces" to germinate. milkoi is in doubt until such structures can be found. In fact, Because zygospores from T. autumnalis lacked the wall some of the commonest species of the Enthomophthorales-for structure, visual appearance of cellular contents, and regular example, Conidiobolus coronata-were long known only from ovoidal shape typical of Coelomomyces, and showed no sign the conidial stages (4). of the dehiscence slit so characteristic of representatives of the The purposes of this paper are (i) to report the discovery of order Blastocladiales, it was essential that the germination the conidial stages of the horsefly fungus, (ii) to present a revised process be followed with care. Hence each of the first five slides description and an emended Latin diagnosis, and (iii) to rename prepared was examined daily. the fungus on the basis of its type host-one of the two previ- RESULTS ously proposed generic names (Coelomomyces) being taxo- nomically incorrect and the other (Pseudocoelomomyces) On the third day, slide no. 2 showed a type of germination that highly misleading. established that this fungus belonged in the Entoniophthorales During a visit to Kiev, one of us (5) recognized that the fungus and not in the Blastocladiales. The zygospore germinated (Figs. described by Dudka et al. (1) was not a Coelomomyces. Par- 2A, 3A) by extruding a through the smaller end of the asitized larvae (see Fig. 1) were duly brought back to North cell (Figs. 2 A-H, 3 B-F). This pattern is altogether distinct America by Laird for germination studies by Couch and Nolan. from that of Coelomomyces, in which sporangial germination Because Dubitskii et al. (2) had described the germination of involves dehiscence through a preformed slit. When the zy- of gospore was floating at or very near the surface of the water, the "sporangia" as involving the production and emergence the stalk of the -bearing structure was short, and the zy- zoospores, it was necessary to see if their observations could be gospore with the four linearly arranged cells (Figs. 2 I and J, confirmed. 3 G-I) strikingly resembled the basidial apparatus of certain rusts, for example Gymnosporangium macropus. When, The publication costs of this article were defrayed in part by page was immersed in 1 or 2 mm of water, charge payment. This article must therefore be hereby marked "ad- however, the zygospore vertisement " in accordance with 18 U. S. C. §1734 solely to indicate the germinating hypha grew to the surface and there formed this fact. the spore-bearing structure. As the hypha grew the protoplasm 2299 Downloaded by guest on September 24, 2021 2300 Botany: Couch et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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Fi(;. 2. Camera lucida drawings. Iy J.N.C. (X985.) (A-F) Early stages in the germination of zygospores; note disappearance of inner wall. (t7-J) Later stages in germination of zygzospore at the water surface. Note conspicuous nuclei (n) in H and conidia (c) in I, maturing in succession fronm the tip. In J all foir conidia have been discharged; note conspicuous vacuole (v) and columella (cm). (K) Primary conidium (p) that has produced a secondary conidium (s). (L) One cell of conidiophore that has discharged its conidium but that failed to get away.

was close to the growing tip, leaving the remainder of the hypha seen; mycelium initially nonseptate, becoming septate at ma- empty. While this growth occurred, septa were formed that turity and filling the larval hemocoel with hyphal bodies that separated the protoplasm from the empty portion of the hypha. give rise to spherical or subspherical gametangia 12-18 prm in This pattern is characteristic of certain Entomophthorales, such diameter, colorless, smooth, with a thin membrane and granular as Conidiobolus, and further study showed that conidial for- contents; gametangia fusing in pairs, one of them emptying its mation and discharge in this fungus resembled that of Coni- contents into the other to form a zygospore. Mature zygospores diobolus brefeldianus Couch (7). It is necessary, therefore, to ovoid, or oval, rarely pyriform; 12-20 X 23-30 pm, rarely up revise the descriptions given by Dudka et al. (1) and Nam and to 40 gm long; colorless, wall smooth, 2-4 pm thick, two- Dubitskii (3). layered; zygospores with finely granular cytoplasm, many small rounded lipoid bodies, a single diploid nucleus and a vacuole. DESCRIPTION Zygospores germinating in water after 3-5 days and sending Tabanomyces milkoi (Dudka arid Koval) emended Couch, out, typically, a linear, four-celled conidiophore (rarely three-, Andreeva, Laird, and Nolan. two-, or one-celled). Conidia formed only in the air at the sur- = Coelomornyces milkoi Dudka and Koval 1973. face of the water or other substrate. Functional conidiophores = fPseudocoelomomyces milkoi Nam and Dubitskii 1977 bearing structures always with a conspicuous vacuole, which (generic name not validly published). persists until after conidial discharge. Conidia forcibly dis- Infection stages and early development of mycelium not charged by the sudden out-pushing of the papilla from inside Downloaded by guest on September 24, 2021 Botany: Couch et al. Proc. Nati. Acad. Sci. USA 76 (1979) 2301

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Fiu.. :3. Photographs. (X418.) (A) Two zygospores, on left quiescent, on right starting germination; note increase in size and thinning of wall. (B--I)) Early stages in germination of zygospores; note marked decrease in thickness of wall. (E) Almost empj)ty zygospore. and nearly mature linear conidiophore. Arrows point to nuclei. (F) Conidiophore beginning to form conidia. Vacuole (v) plays an imuportant role in conidial dischare. Seven zygosl)ores that have not started to germinate. (U and H) Stages in coniidial formation. (I) Conid(iophore with all four conidia shot away. Note conspicuous vacuoles in zygospore and conidiophore. Photographs by ,J.N.C.

the conidium (Fig. 2L) with such force that they are shot away conidium, which is shot off in the same manner (Fig. 2K). This from the columella for a distance of several hundred micro- process may be repeated several times, as in Conidiobolus meters. If a conidium fails to germinate, it may form another brefeldianus (7). Downloaded by guest on September 24, 2021 ~~~~~Proc.23022302Botanyl~~~otany:CouchCouchetetal.al.Nati. Acad. Sci. USA 76(1979) DIAGNOSIS prominent among such carriers of the latter disease, and at least four are the vectors of loiasis the filarial worm Thbanomnyces genus novum. species (caused by Mycelium maturumt primo haud septatum, denique sep- Loa loa) in West Africa (9). The of a of the tatum, in corpora hyphalia sectum, haemocoeliam vermiculi discovery representative Entomophthorales, complens; corpora hyphalia gametas sphericas vel subsphericas the zygospores of which germinate to form a linear, four-celled, gignentia; ganmetae in paribus conjunctae, alia in aliam fusa, spore-bearing structure, is significant. The most common pat- Zygotam zygosporae ovoideae, ovales, vel tern of zygospore germination known to occur in the Ento- efformantes; pyri- involves the of a fornies, hyalinae, crasse tunicatae, plasmate granulari et mophthorales production nonseptate germ vacuolo impletae, uninucleatae, in aquo germinantes, coni- tube, which in turn produces a solitary terminal germ conidium diophorum lineare, plerumque quadricellulare, instar Ure- [for example, Entomorphthora egressa; (10)1. The production dinurhi promycelii emittentes; conidia, in aere tantum emissa, of a septate germ tube with a terminal conidium and one or eversione papillae basalis per vim propulsa, nonnumquam per more lateral conidia has been reported for refpetitionem germinantes. aphidis by Tyrrell and MacLeod (11). Type: Tabanomyces milkoi Couch, Laird, Andreeva, and The latter authors, however, reported that the total number Nolan. Type deposited in the herbarium of the Mycological of germ conidia on a germ tube was most commonly two and Institute of Botany, Academy of Sciences, Kiev, Ukraine, USSR; occasionally three. Because of the lack of cytological informa- and the Mycological Herbarium, University of North Carolina, tion for E. aphidis, only the similarity in germ tube structure Chapel [li, NC. and the partial similarity in germ conidium number and placement can be noted. The pattern of development in T. DISCUSSION milkoi is strikingly similar to that of teleutospore germination In the formation, structure, and size of the zygospores this to form a promycelium and bagidiospores in the rusts. Thus, the species resembles Entomophthora fresenii as described by presence of this basidium-like structure in T. milkoi reinforces Tlhaxter (4). Entomophthora fresenii, which possesses elliptical the old concept that the higher fungi may be closely related to zygospores, is the only known species in the Entomophthorales the Entomophthorales. that does not have spherical or subspherical zygospores (4, 8). Because of the similarity in manner of formation and the Also, in the Entomophthorales the zygospores usually have one structure of the zygospores of E. fresenii and T. milkoi, and in large lipoid body, whereas, in E. fresenii and the present the zygospore germination patterns of T. milkoi and E. aphidis, species, the zygospores contain numerous small lipoid bodies. it is of interest to note that the pattern of conidium germination In F. fresenii, according to Thaxter (4), the zygospores are is similar for E. aphidis (11) and E. fresenii (12). formed by the conjugation of two small, spherical hyphal bodies Above the of contact of the bodies, Thanks are due to Yola Hurwitz of Memorial University's Research (gametangia). region hyphal Unit on Vector for the Russian arises as a bud. the wall Pathology translating papers (refs. 1-3), the zygospore During conjugation to Dr. D. P. Rogers of the Department of Botany, University of Illinois, around the two gametangia thickens. Portions of the two walls Urbana, IL, for providing the Latin diagnosis, and to Dr. L. S. Olive remain attached to one of the ends of the elliptical zygospore of the Department of Botany, University of North Carolina, Chapel as bladder-like appendages that are generally persistent. Hill, NC", for valuable suggestions. This study was supported by the In T'. milkoi, the conjugation of two spherical gametangia National Research Council of Canada. to form the zygospore has been observed by one of us (An- Ibut further studies are needed on the of the 1. Dudka, I. A., Koval, E. Z. & Andreeva, R. V. (1973) Novitates dreeva); origin Systena ticae Plan tarurn non Vasculariunn 10, 88-91. gamnetanigia, their structure, and the conjugation process, to 2. Dubitskii, A. M., Deshevigh, N. D. & Andreeva, R. V. (1974) determine if conjugation in the present species is similar to that Parazitologia 8, 175-178. described] for E. fresenii by Thaxter (4). It would be interesting 3. Nam, E. A. & Dubitskii, A. M. (1977) A Review of the Systematic to see if the zygospores of F. fresenii germinate in the same Status of the Fungus Coelornornyces milkoi and a Description nmanner as in T. milkoi. Oval conidia formed sparingly on of a New Genus of the Entomophthorous Fungi Pseudocoelo- slender conidiophores have been described by Thaxter (4) for momyces (Entomophthorales), VINITI No. 1309-77 Dep., UDK E. fresenii. 591.69-577.1:576 (Acad. Sci. Kazakh. SSR, Inst. Zool., Alma Ata, The zygospore in Tabanomyces is formed by copulation of USSR). twov( uninucleate gametangia. Nam and Dubitskii (3) and one 4. Thiaxter, R. (1888) Mern. Boston Soc. Nati. Hist. 4, 133 201 of us (Andreeva) observed nuclear fusion and the formation of (Plates 14-21). We have observed that the mature 5. Laird, M. (1976) "Report on a visit to the USSR, concerning unihuctileated zygosp~ores. 1)iocontrol of vectors (18 April-10 May 1976)" (Research Unit ZVgospore is uininuicleated anid that during germination the on Vector Pathology, Memorial Universitv of Newfoundland, diploid nucleus (livides and then divides a second time. Thus, St. John's, NF, Canada). foutr nuclei enter the conidiophore (Figs. 2H, 3E), and one 6. Couch, J. N. (1967) in Proceedings of the joint United States- nucleus migrates into each of the four . Chromosome Japan Seminar on Microbial Control of Pests, Fukuoka, counts have not beeii conducted; but if, as we believe, these are Japan (Institute of Biological Control, Faculty of Agriculture, ineiotic (livisions, the conidia must result from a sexual process. Kvushu University, Kvushu, Japan), pp. 93-105. They coutldl, therefore, be called meioconidia, not simply con- 7.Couch, J. N. (1939) Am. J. Bot. 26, 119-120. idia (which are asexual propagules). 8. MacLeod, D. M. (1963) in Insect Pathology. An Advanced These meioconiclia are the m-ost likely agents for the infection Treatise, ed. Steiphaus, E. A. (Academic, Newv York), Vol. 2, pp. o)f tabanid larvae. However, lbecause the observed level of zy- 189-231. 9. Smith, K. C. V., ed. (1973) and Other Arthropods of gospore germination under laboratory conditions is only about Mledical Importance (Br. Muns. Nat. Hist., London). 5%, this lo)w rate would need to be greatly increased in order 10. Nolan, R. A., Dunphy, C. B. & MacLeod, D. M. (1976) Can.]J. to give T'. inilkoi practical biocontrol potential against Taba- Bot. 54, 1131-1134. nidlae. This family of biting flies includes severe pests of man, 11. Ty'rrell, D. & MacLeod, D. NI. (1975) Can.]J. Bot. 53, 1188- domestic animals, and wildlife, particularly in boreal regions, 1191. as well as mechanical carriers of certain zoonoses, including 12. Soper, 11. S. & MacLeod, D. M. (1963) J. Insect Pathol. 5, surra and tularaemia. Deerflies of the genuis Chrysops are 4718-482. Downloaded by guest on September 24, 2021