DISCOVERY OF ASCOMYCETE CHARACTERISTICS IN SPORO- THRIX SCHENCKII

M. Thibaut and M. Ansel

Sporothrix schenckii has been stuãied by light microscopy, and also by transmission and scanning electron microscopy. Characteristics of Ascomycetes have been oibserveã at the levei of the cell-wall anã in the synaptic system of the hyphae. Also the perfect state has been discovereã. The four spored asei are formed directly from the mycelium and there is no fructification. Doli- choascus schenckii is the name suggested for this perfect state which constitutes a new of the Endomycetaceae.

INTRODUCTION tion of the lesions in animais we studied the cytology of the . In light mi­ Sporothrix schenckii is a fungus that croscopy, we went carefully over the clas- can be a parasite on man or animais as sical cytologic and histologic examinations. well as a saprophyte on dead plants. It In electron microscopy, the specimens were can be found in different states: either prepared acoording to methods previously in the saprophytic mycelial phase corres- described by Thibaut (8). They were ob- ponding to its growth in nature and in served first with the transmission elec­ culture, or in the — like cells ob- tron microscope (Hitachi HU 11 B, Sie­ tained in complex cultural conditions cor- mens Elmiskop 1, Jem 100 U and Jem 100 responding to the parasitic state. Until B Jeol) and secondly with the scanning now considered as a Deuteromycete, Spo­ electron microscope (JSM U 3 Jeol). rothrix schenckii owed its generic name and place in classification to the charac­ RESULTS teristics of its asexual reproduetion which was then the only one known. Our expe- The cultures of mycelial form on Sa: riments have revealed ascomycete charac­ bouraud’s glucose agar are more irregular teristics and the existence of sexual re­ in the center than on the edges. Their produetion. colour is more or less ochre (fig. 1). Mi- MATERIAL AND TECHNIQUES croscopic examination shows a very fine creeping mycelium, septate, intertangled Our research was practised on a strain and branching out. From it grow conidias of Sporothrix schenckii, preserved in the or asexual spores, the only ones discovered mycotheque of the parasitology laboratory so far; these are either clustered around of the Medicai school of the University of and along filaments and inserted by means Paris. It had been isolated in 1942 in a of sterigmates, or clustered at the extre- South-African patient suffering from spo- mities of recumbant filaments where they rotrichosis. After experimental reprodue­ form a characteristic bouquet (fig. 2).

Laboratoire de Parasitologie, 15 rue 1’Ecole de Médecine, 75 — Paris, VI, France Submitted to publication on 11.20.: 2. Rev. Soc. Bras. Med. Trop. Vol. VII — N? 6

The mycelial form grown in Mariats mycelium (fig. 9 and 10). Each of the shaken infusion, in anaerobiosis at 37°C, coupled hyphae has a bulging head taking changes into yeast - like cells (fig. 3). In up 25% of their height . These heads grow an animal such as a mouse, inoculated close to each other and seem to unite. Very with sporotrichosic pus or a culture of likely these hyphae are sexual organs Sporothrix schenckii, a parasitic and very between which there is copulation such as polymorphic form is otaserved in the le- can be observed among the Endomyceta- sions, but the most characteristic feature ceae. is the cigar shaped body (fig. 4). Excep- Examination by transmission electron tionally, among such rodents as the hams­ microscopy of ■section cut from cultures of ter, asteroid bodies can be seen (fig. 5). old yeast cells proved to be confirmation Study in transmission electron micros- of the existence of sexual reproduction. copy of the hyphae of Sporothrix schenckii Indeed we were able to discover (1) typical first showed us the existence in the cell- asei and ascospores (fig. 11 and 12). walls and septa of particular structures The mature ascus is oblong-shaped with common to Ascomycetes and red algae, thinning extremities. It is 5 n long and with Woronin bodies around the septa; it 2,5 n wide. It normally contains four as­ secondly showed the presence of the same cospores, slightly more elongated, 3 ^ in synaptic system between the cells as that lenght and 1 /i in width. There is no of the Ascomycetes. In the lateral cell- fruetification. According to the incidencce walls (fig. 6), going from outside to inside, of the section, the appearance of the ascus we found the following formations, named may vary, as may the number of visible after Chadefaud’s terminology (4): a tu­ ascospores. bular vagina enveloping the hypha; a Since the discovery of asei and ascos­ sub-vagina; a film lining the inside of the pores the species Sporothrix schenckii can sub-vagina; a locula encasing each of the no longer be considered as a Deuteromy. articles contained in the sub-vagina. cete, but it must now be definitely con- Between the successive articles of the hy­ nected to the class of the Ascomycetes. phae the septum is formed by the extre- The well developed mycelium, the absence mities of the loculae of the two articles of fructification, the formation of the ascus separated by the septum and interlocular through the copulation of two particular system placed between the two loculae. hyphae should lead us to classify it in the The septum of Sporothrix schenckii has family of the Endomycetaceae. But in this a central hole, but when the lengthwise family we did not find any speccies to section does not pass the levei of the pore, which it could be related. On account of the septum appears to be continuous. At its four ascospores and its biology, it seems the pore levei there is a complex synaptic nearer the Eremascus genus. However it system (fig. 7). The pore is covered on has neither globulous ascospores nor thick each side with a dark lenticular disc, thinn- mycelium. Considering the very fine my­ er on its edges. Between the discs and celium of Sporothrix schenckii (1 n wide perpendicularly to them, thin trabecules and often less), its characteristics vege­ can be seen. They seem to pass through tative multiplication system, and, most of the discs and radiate in a short fan- ali, the obviously elongated shape of its shaped spray outside the discs and into asei and ascospores, the species schenckii the cytoplasm of each one of the adjoin- appears to us as a new type of fungus, ing articles. The plasmalemma surrounds whose name we have suggested to be: Do- the border of the pore and insinuates itself lichoascus Thibaut and Ansel 1970. between this border and that of each of the two discs. On either side of the cen­ DISCUSSION tral synaptic system we observed Woronin bodies, generaly two or three in number Chadefaud’s experiments in optical mi­ on each side of the septum and around the croscopy detected that the cell-walls of pore (fig. 7 and 8). ascomycete hyphae include two layers: the Observation of the mycelial form in externai one called the vagina of the hy­ scanning electron microscopy, enabled us pha, and the internai one formed by the to discover a numbgr of notable pairs of loculae of the cells. Again we discovered filaments standing above the vegetative these elements within the ultrastrueture Nov.-Dez., 1973 Rev. Soe. Bras. Med. Trop. 343 of the mycelial filaments of Sporothrix intra-hyphal hyphae like those Carbonnel schenckii. The various layers of the fungai and Rodriguez (3) observed in Paracocci- cell-wall described by Chadefaud (4) are dioides brasiliensis. of course much more evidently visible in Our observations agree with those of electron microscopy. Schrantz (7) made Vuillemin (10) who, on the subject of the same observations in the cell walls of the crateriformis studied by vegetative hyphae among the Pustularia Hudelo, Sartory and Montlaur (5), which cupularis and the Gálactinia plebeia As- is a pathogenic species discovered in a comycetes. cutaneous lesions in man, found that the The septal perforations were studied in process of asexual reproduetion of this pa­ electron microscopy by several botanists: rasites placed it — from the vegetative Carroll (3) with Saccóbolus and Ascodes- point of view — among the Sporotrichum mis, Schrantz (7) with Galactinia plebleia (which, to-day, we should call Sporothrix.). observed synaptic systems offering many In the reverse — we ourselves were led to relationships with Sporothrix schenckii. consider the perfect atate of Sporothrix On the other hand these men showed that schenckii as an Endomycetaceae. the Woronin bodies, also observed by us, are typical of the Ascomycetes only. CONCLUSION. Could the perfect state of Sporothrix schenckii be interpreted differently? It Our research on Sporothrix schenckii could not be confused with that of endo- has shown cytologic characteristics of As­ conidia, such as Saez (6) described them comycetes at the levei of the cell wall, the in the genus, for these are septa and the synaptic system of the hy- formed in single files, one after another pliae and, besides, a perfect state of sexual and are not grouped like ascospores. Indeed reproduetion unknown until now. There- there are endospores in old cultures of fore our experiments bring new taxonomic Sporothrix schenckii, but they are very elements which change the category of different from ascospores and cannot pos- this species completely, and it can new be sible be confused (1). Nor could they be classified with the Endomycetaceae.

RESUMO

Sporothrix schenckii foi estudado em microscopia óptica e em microscopia eletrônica. Foram observados caracteres citológicos de Ascomicetos nas pare­ des das células e no aparelho sináptico dos hyphae. Foi descoberta a reprodu­ ção sexuada. Os ascos de quatro esporos são formados diretamente sobre o micélio e não há frutificação. Dolichoascus schenckii é o nome proposto para este estádio perfeito que constitui um novo gênero de Endomycetaceae.

REFERENCES

1. ANSEL, M. & THIBAUT, M. — Une croscope study J. Bacterior., 96: 533- nouvelle Endomycetaceae: Dolichoas­ 543, 1968. cus nov. gen. Découverte de la repro­ duetion sexuée par asques chez le 3. CARROLL, G. — The fine strueture Sporotrichum schenckii (Hetkoen et of the ascus septum in Ascodesmis Parkins 1900). C.R. Acad, Sei., 270: sphaerospora and Saccobolus kerverni. 2171-1173, 1970. Mycologia, 59: 257-532, 1967. 4. CHADEFAUD, M . — Les végétaux 2. CARBONNEL, L. M. & RODRI- non vasculaires. Tome I du Traité de GUEZ, J . — Mycelial phases of Para- Botanique systématique de M.CHA- coccidioides brasiliensis and Blasto- DEFAUD et L. EMBERGER. Masson, myces dermatitidis: an electron mi­ Paris, 1960. 344 Rev. Soc. Bras. Med. Trop. Vol. VII — N9 6

5. HUDELO, SARTORY et MONTLAUR. mique du Sporotrichum schenckii — cité par VUILLEMIN, p. 1930 in (Hetkoen et Perkins) de Beurmann Les champignons parasites et les my- et Gougerot: Dolichoascus schenckii coses de l’homme. Lechevalier éd. (H et P) Thibaut et Ansel, 1970. These 1930. de Doctorat d’Etat ès Sciences, Paris, J970. 6. SAEZ, H. — Formation d ’endospores chez Geotrichum candidum. Ann. P a­ rasitai. hum. comp. 44: 197-204, 1969. 9. THIBAUT, M. & ANSEL, M. — Ob- servations microscopiques sur les phé- 7. SCHRANTZ, J. P. — Etude cytologi- nomènes de senescence chez le Sporo­ que en microscopie optique et electro- thrix schenckii. Ann. Parasitol. hum. nique de quelques Ascomycetes. II. comp. sous presse, 1972. La paroi. Rev. Cytol. Biol. veg., 33: 111-156, 1970. 10. VUILLEMIN, P. — Les champignons 8. THIBAUT, M. — Etude biologique, parasites et les mycoses de l’homme. morphologique, cytologique et taxino- Lechevalier, Paris, 1931.

Fig. 1 — Microscopíc view of Sporothrix schenckii on Sabouraud’s glu­ cose agar.

Fig. 2 — Microscopic view of the mycelial phase. Bouquets of conidia. Coloration: methyl blue.

Fig. 3 Microscopic view of the yeart — like cells. Coloration: methyl green.

Fig. 4 Parasitic phase in vivo: cigar — shaped bodies in pus (mouse) Coloration: carbolic thionine.

Fig. 5 Asteroid body in tissues (Hamster) . Coloration: Hematoxylin and eosine.

Fig. 6 Various constructive layers of the cell wall and septum on a hypha of Sporothrix schenckii (longitudinal section) . Fixatlon: glutaraldehyde and osmium tetroxyde. Contrastation: uranyl acetate and lead citrate. Abbreviations: P: Plasmalemma; I.S.: interlocular strip; I.R.: interlocular ring; L: locula; V: vagina; S.V.: sub vagina; F; internai film lining sub vagina.

Fig. 7 - Longitudinal section of a mycelial filament showing — on the levei of septal pore, — the synaptic system composed of two discs crossed by trabecules. Four Woronin bodies can be seen near by. Fixation: glutaraldehyde and osmium tetroxyde. Contrastation: uranyl acetate and lead citrate. Abreviations: L: lomasome; L.B.: lipid body; W.B.: Woronin body; P.: plasmalemma; S.: septum; D.: disc.

Fig. 8 - Longitudinal section in a hypha on the levei of septum with no pore. Note two Woronin podies. Fixation: glutaraldehyde and osmium tetroxide. Contrastation: uranyl acetate and lead citrato. Abbreviations: W.B.: Woronin bodies; P.: plasmalemma; S.: septum; V.: vacuole.

Fig. 9 - Sexual filaments of Sporothrix schenckii. Fixation: formalin. Scanning electron microscopy.

Fig. 10 ■ Sporothrix schenckii, pair of sexual filaments. Fixation: formalin. Scanning electron microscopy.

Fig. 12 Section of an immature ascus. Fixation: glutaraldehyde and osmium tetroxide. Contrastation: uranyl acetate and lead citrate.

Fig. 12 — Mature ascus with three well differentiated ascospores. Fixation: glutaraldehyde and osmitmi tetroxyde. Contrastation•. uranyl acetate and lead citrate.