Dehiscence and active spore release in pathogenic strains of the ...

Dehiscence and active spore release in pathogenic strains of the yeast Metschnikowia bicuspidata var. australis: possible predatory implication MARC-ANDRE LACHANCE, M A R YMIRANDA, MARTINW. MILLER,A N D HERMANJ. PHAFF De/~trr/rtror/c!f'Footl Scirrrc.r rrrrrl Teclr~rolo~yy. Llrri~~c,rsrr~ of Ctrlij&~rzitr, Dcr1,is.C A , U . S . A . 95616

Accepted September 14. 1976 LACHANCE. M-A,. M. M I R A N D A M., W. M I L L E Rand . H . J . PHAFF.1976. Dehiscence and active Dicirspitltrrtr var. crr~srr~ctlis: spore release in pathogenic strains of the yeast Mei.scl~r~ikon~itr possible predatol-y implication. Can. J. Microbiol. 22: 17561761. Strains of Mc~!.sclrriil;o~~~icr bicrr~piclt~itrvar. r~rr.srrolis.pathogenic to brine shrimp (Ar.io,zicr srrlirro). were observed to form asci which, upon reaching maturity, forcibly expelled their needle-shaped spores. The mechanical fol-ce responsihle apparently originates from the formation of an ectoplasmic mucilage capable of exerting pressure over all of the ascus contents; when the apex of the peduncle I.ilptlll.es, the ascospores are violently released. Cytochemical analyses indicated that the gel is a substance highly resistant to chemical and enzymatic hydrolysis. Its chemical nature is not known as yet. The mo~phogeneticevents of this processare described, and its ecological implication, the possibility of active mechanical predation in yeast, is discussed. L A C H A N C M-A,, E, M. M I R A N D A M., W. M I L L E R et H . J . PHAFF.1976. Dehiscence and active ~~~i(~ var. L I I I S ~ I Y I ~ ~ S : spore release in pathogenic strains of the yeast M ~ i s ~ l r r r i k o bicrispidtri(r possible predatory implication. Can. J. Microbiol. 22: 17561761. Nous avons observe, chez des souches pathogenes (pour Arienrio solirrcr) d e Itlctsc~lrtriko~~~icr hicrt.spidoio var. rrrr.sirtr1i.s.la formation d'asques capables. h maturite. d'exp~~lseravec force leurs spores en forme d'aiguille. Les forces mecaniques responsables sont apparemment causees par la formation d'un mucil;~geectoplasmiq~resource d e pression interne. L'ouverture soudaine de I'extremite du pedoncule permet un rel%chement subit de cette pression, causant en mPme temps la liberiltion violente des spores. L'analyse cytochimique revele que le gel en question resiste B I'hydrolyse chimique et enzymatique. Sa composition n'a pas encore ete elucidke. Nous decrivons les evenements morphogenetiqi~esdece phenomene et discutons la possibilitt qu'il s'agisse d'un cas de predation active chez les lewres.

Introduction Internal parasitism of plants and animals is known among species of the three yeast genera Metscht~ikowia,Netmztospora, and Coccidiascus belonging to the Spermophtoraceae, in which the ascospores are in the form of sharp-ended spindles, or needles (Lodder 1970). This feature allows the spores to penetrate the eventual hosts more easily, as a result of their small crosssectional diameter. Yeast of the genus Metscht7ikowia were first isolated from diseased Duphrlin inngr7n by bicauspidata Metschnikoff (1 884). Metscht~ikow~irr was described by him as a parasite of this crustacean; its free needle-shaped spores were observed piercing the intestinal lining of the animals. Kamienski (1899) isolated another species (M. artenfine) from brine shrimp (Artemin salilln) and reported that its spores were liberated from the asci by a process which involved the swelling of a mucilage that originated from the protoplasm. This was the first published observation

of naturally occurring dehiscence in M e t s c k ~ ~ i koivin. Subsequent infections attributed t o this yeast have been reported for Daphnia (Chatton 1907; van Uden and Castelo-Branco 196 I), Dnsj~llelea (Keilin 1920), Artetuia (Spencer et a/. 1964), a n d Calat~us(Seki and Fulton 1969). Miller et a[. (1967), Pitt and Miller (19700, 1970b), and Talens et nl. (1973) have studied various aspects of sporulation in the species of Metscht~iko~jia, using light a n d electron microscopy. They were successful in freeing the spores by mechanical (micromanipulation) or enzymatic (snail gut juice) methods; natural dehiscence, however, was only observed infrequently , for some strains of M. p ~ i l c l l ~ r r i na~ aterrestrial, lion-pathogenic species (Pitt and Miller 1968); 110 description of the process, nor the mention of a violent discharge were given. The present paper reports light-microscopic observations of ascal lysis and active spore release in strains of M. bicuspicklta var. a~rstralis parasitic t o brine shrimp. The mechanism of

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discharge is discussed along with its ecological significance.

homogeneous, continuous layer of ectoplasm was observed around the spores, the vacuole, and the epiplasm. Materials and Methods Illustrated in Fig. 1 are asci and spores a t Yeast Crtltrrres different stages of the dehiscence process. First, A sample of commercially reared A. snlir7a had been the apical portion of the peduncle wall underbrought to this laboratory for examination of a possible infection of the brine shrimp by the yeast Metschniko~via. goes hydrolysis, as evidenced by the appearance of dense rings ( a , 6). Then, a small evagination Microscopic observation confirmed the presence of yeast in the body cavities of diseased shrimp, and three strains of protoplasm progressively swells ( c ) until it were isolated in pure culture. They were deposited in the suddenly collapses; a t this point the spores are yeast culture collection of the Department of Food ejected along with the epiplasm and the vacuole Science and Technology, University of California, Davis, (d, e), leaving a seemingly empty ascus ( I ) ; under the numbers: 76-41, 76-42, 76-43. Identification as M . bicrrspidata var. australis was obtained following liberated ascospores ( g ) are found outside the the criteria of Miller and van Uden (1970). ascus. Part of this sequence of events was followed in an individual ascus, as depicted in Microscopy Mounts were prepared by placing individual shrimps, Fig. 2. The apex of the peduncle is digested (a), at various stages of infection, on glass slides coated with leading t o the protrusion of some intracellular a thin layer of 1% agar (Difco, Detroit, MI); a cover slip material still bound by the cytoplasmic memwas placed over the material and sealed with vaspar. brane and some residual wall material (b). This The specimens were observed with a Nikon M35S bulge increases in size (c), and then rapidly Apophot Microscope, under phase-contrast illumination, and the observations were recorded on Panatomic-X regresses (probably after the wall material has film (Eastman Kodak, Rochester, NY). reached a critical degree of fragility), at which point the ascus contents are violently expelled Cytochen~icalMethods Preparations of asci were digested with the following (d). Steps a to c took place over a period of 2 h, enzyme preparations: a-amylase (Sigma, St-Louis, MO); whereas the time between the collapse of the chitinase (Nutritional Biochemicals, Cleveland, OH); glusulase (Endo, Graden, NY); lysozyme (Calbiochem, evagination and the ejection was very short, San Diego, CA); pepsin (Sigma); pronase and pancreatic i.e. 1 or 2 s. These rapidly occurring events are ribonuclease (Calbiochen~);zymolyase (Kirin Brewery, drawn schematically in Fig. 3. Takasaki, Japan). In some asci, the spore expulsion took as Carbohydrates were stained with the following dyes: long as about 5 s and in others, the release was 1% iodine - 2% potassium iodide; 1% night blue (Allied so sudden that it could not be followed by the Chemicals, New York, NY) in 5% ethanol; Schiff's eye. As a result of such a violent ejection, the reagent, prepared as recommended by the Biological Stain Commision (1960). Fats were stained with 1% ascus sometimes recoiled slightly in reaction to Sudan black B (Matheson, Coleman and Bell, E. Rutherthe movement of the ejected materials. ford, NJ) in 7 0 z ethanol. Coomassie brilliant blue (K and K , Irvine, CA) at a concentration of 2% in 7% acetic acid was used to stain protein. The asci used in cytochemical studies were prepared in the following manner: diseased shrimp were crushed in artificial seawater and held overnight. The suspension was then centrifuged at 2000 g for 5 min, and the supernatant, containing bacteria and debris, was replaced with fresh seawater. The washed suspension was then mounted with a n equal amount of enzyme solution or stain.

Results Dehiscetzce and Spore Discharge Microscopic mounts of infected brine shrimp contained numerous club-shaped cells. After incubation of the prepared slides at room temperature overnight, most asci had reached maturity. They typically contained two ascospores, tightly juxtaposed, and surrounded by dark granules. A large vacuole filled a significant portion of the wide end of most asci, and a thick,

Mechanist~lof Spore Discharge It was first thought that the mechanism by which the spores are expelled was of a plasmolytic nature, where the amorphous peripheral zone of the cytoplasm would be interpreted as an enlargement of the periplasm. However, a close examination of 'empty' asci indicated that only in the first third o r half of the peduncle was there a space in which free movement of particles, such as bacteria o r India Ink, could occur. Kamienski's (1899) description of the dehiscence referred to the possible involvement of a mucilaginous substance developing from the inside of the plasmalemma. Our observation that Brownian movement is lacking in the dehisced asci supports this viewpoint. T o obtain further knowledge o n this mucilage, washed suspensions of asci a t various stages of maturity

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C A N . J. MICROBIOL. VOL. 22. 1976

Frc. I . Asci and ascospores of IM.bicrrspirln/a var. alw/r.cilis at different stages of the dehiscence process. Mag. n, 6, c, f: 1500 x ; cl, e : 1800 x ; g: 1000 x . ( u = vacuole; ec = ectoplasm; r. = wall lysis rings).

FIG.2. The dehiscence process in an ascus of M . bicrispicfci~c~ var. nfts/~.olis.Mag. (I, 6, c : ZOO0 cl: 1300 x . Time between observations: 0-6, 2 11; 6-c, 10 min; c-cl, 10 s.

were digested wit11 different enzyme preparations. Glusulase and zy~nolyase(both of which have lytic activity on cell walls and ascus walls of yeast), a t final concentrations o f a b o u t

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0.1 ml/ml a n d 0.5 n~g/nll, respectively, were found effective in hydrolysing the ascus walls. It was noted that, in asci t h a t had already dehisced, the tendency was for the early (10-

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FIG.3 . Forcible discharge of ascospores by an ascus of M. bicrlspi(/otn vat. n~rstrcrlis.Mag. 1500

15 min) lysis to be preferentially at the swollen part of the ascus (Fig. 4a). This was also the case for asci at an advanced stage of development. Conversely, immature asci were more resistant to hydrolysis and showed weakening of the wall a t randomly distributed areas around the ascus, and only after a longer period of incubation (45 min, Fig. 46). Part of the digestion process of a dehisced ascus by zymolyase was photographed at intervals, as shown in Fig. 5. The mucilage remained intact, even after 8 h of digestion. This indicated that its nature is different from that of the P-(1 -* 3)-glucan component of the cell

x

wall. PI-eparations of asci that had undergone digestion by zymolyase for at least 20 min were subjected to digestion by various other enzyme preparations, in the hope to obtain clues as to the chemical nature of this mucilaginous material. a-Amylase, chitinase, lysozyme, pepsin, pronase, and ribonuclease, all a t final concentrations of 0.5 mglml, were ineffective, after 6 h, in liydrolysing the material, although the effects of their activities were often evident on other materials present in the preparations. Digested asci were also stained with iodine potassium iodide, Schiff's reagent, night blue, Sudan black B, and Coo~nassiebrilliant blue;

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CAN. J MICROBIOL. VOL 22, 1976

FIG.4. Asci of M. bicrrspidata var. arrstralis after digestion with zymolase. (a) Dehisced asci, 1800 x ; (b) immature ascus (lysed by enzyme treatment), 2000 x . all stains visibly reacted with some materials, but failed t o stain the mucilage. Similar preparations were also treated a t room temperature with HCI and NaOH, at a final concentration of about 2 N. Only the latter seemed to produce a partial distortion of the shape of the gel, but both failed to dissolve it. Finally, another digested preparation was heated until it boiled, with little o r n o visible effect o n the substance. Its nature, at this time, is thus unknown.

FIG.5. Enzymatic digestion of the wall of a dehisced ascus of M. bicuspihm var, nlrstrnlis with zymolyase. Digestion times: 6 min (a); 12 rnin (b); 24 min (c); 36 rnin (0).Mag. 1500 x .

Discussion Evidence has been presented for the first time, substantiating the existence of an active predatory mechanism in yeast. Metschnikoff (1884) had advanced the concept that the aciculate shape of the ascospores of Metschfzikowia allowed them to penetrate more easily the intestinal lining of 'prey.' This structural feature is also present in the yeasts Nenzatosporn a n d

LACHANC:E ET AL.

Coccidiascus, both known to be internal parasites of higher organisms (Lodder 1970). The observations reported here make it conceivable that Metschnikowia not only possesses, by virtue of its spore morphology, passive infective features, but can also actively attack individuals by its forcible spore discharge. This would presumably involve immediate penetration of discharged ascospores into vital elements of the crustacean's bodv. For this to be verified. an ability for the asci to affix themselves internally or externally to some part of the shrimp will have to be demonstrated. As to the mechanism of forcible spore discharge, it appears that, under suitable conditions, an enzymatic process leads to the formation of a highly hydrophilic gel in the ectoplasmic region of the ascus, resulting in the buildup of high internal pressure. Localized hydrolysis of the apical wall of the peduncle allows this pressure to be dissipated rapidly, simultaneously enabling the spores to be expelled violently. This may provide clues to explaining other poorly understood forms of forcible spore discharge in yeast. Failure to obtain hydrolysis of the mucilage with four different kinds of carbohydrases, two proteases, ribonuclease, a strong acid, and a strong alkali, or heat, indicates that this gel is of a highly resistant nature; in addition, the absence of reactions to a fat stain, a protein stain, and three carbohydrate stains, covering a wide spectrum, leaves its identity unknown. Production and isolation of substantial amounts of the ectoplasmic gel will be necessary for its chemical analysis. The only remote clue to the composition of this substance is the observation that the thickness of the ectoplasmic layer is inversely proportional to the resistance of the adjacent wall to enzyme digestion, suggesting that endogenous hydrolysis products of glucan might be used in the synthesis of the mucilage.

Acknowledgements One of us (M.-A. Lachance) acknowledges the awarding of a graduate scholarship by the National Research Council of Canada. We are

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greatly indebted to Professors Kenneth Wells and Herbert B. Currier of the Department of Botany, University of California, Davis, for helpful discussions and suggestions. Our gratitude is also extended to the Kirin Brewery, Takasaki, Japan, for the samples of zymolyase. BIOLOGICAL S T A I NCOMMISSION. 1960. Staining procedures. The Williams & Wilkins Company, Baltimore, Md. CHATTON,6. 1907. Revue des parasites et des commensaux des Cladockres. Obsel-vations sur les formes nouvelles ou peu connues. C. R. Assoc. Fr. Avanc. Sci. Congrkb de Reims. 797-9 1 1. K A M I E N S KTH. I , 1899. Notice prtliminaire s u r la nouvelle espkce de Met.sclrrtiko~~,i(r(Motlospor.tr Metschn.). T I X VSoc. . Imp. Natural. S. Pet. 30: 363-364. K E I L I ND. . 1920. On a new sacch:u.omycete Morro.sporellcr rrt~ictr.spidn~tr gen. n. nom., n. sp.. ~EII-asitic in the body cavity of a dipterous larva (Dtrs~lrcletrohscrrrtr Winnertz). Parasitology, 12: 83-91. LODDER,J . (E(1ifor) 1970. The yeasts. a taxonomic study. North-Holland Publishing Co., Amsterdam. E. 1884. Ueber eine Sprosspilzkrankeit METSCHNIKOFF, des Daphnien. Beitrag zur Lehre iiber den Kampf der Phagocvten gegen Krankheitserreger. Arch. Pathol. Anat. Physiol. Lin. Med. 96: 178-195. M I L L E R ,M . W.. E. R. BARKER,and J . I. PITT. 1967. J . Bacteriol. 94: Ascospore numbers in M(~tsc~Irtriko~~~icr. 258-259. M I L L E R .M. W., and N. V A N U D E N . 1970. The genus Mctsclrtziko~~~itr Kamienski. 111 The yeasts, 21 titxonomic study. Eclit~rlby J. Lodder North-Holland Publishing Co., Amsterdam. PITT,J. I., and M. W. MILLER.1968. Sporulation in Cntlrliclo prrlclrrrvi117o. Corrdido rrrlktrrtjii, and Clrlnt~~ytloZ!I?I(I species: their relationships to Met.sch?rikun~icr. Mycologia. 60: 663-685. 1970n. Speciation in the genus Met.sclrt~iko~~~icr. Antonie van Leeuwenhoek; J. Microbiol. Serol. 36: 357-38 1. -1970h. The parasexual cycle in yeasts of the genus M c ~ t ~ c I ~ t ~ i Mycologia, k ~ ~ ~ ~ i t i 62: . 462-473. S E K I , H., and J . FULTON. 1969. Infection of marine copepotls by Metschrliko117irr sp. Mycopathol. Mycol. Appl. 38: 61-70. S P E N C E R JF.. T..H. J. PHAFF,and N. R. G A R D N E R1964. . Me/.sclrr~iko~~~io ktrtni~trskii, sp. n., a yeast associated with brine shrimp. J. Bacteriol. 88: 758-762. T A L E N SL. , T.. M. W. M I L L E Rand , M. M I R A N D A1973. . Electron micrograph study of the asci and ascospores of MetscIrt~iku\~~ia Kamienski. J. Bacteriol. 115: 316322. V A N U D E N , N . , and R. CASTELO-BRANCO. 1961. Me/sclrt~iko\~~icr ~ o b e l l i si p . nov. and M . krissii sp. nov., two yeasts from the Pacific Ocean pathogenic for Dtrph11ic1t??ogtltr.J . Gen. Microbiol. 26: 141-148.