METSCHNIKOWIA KAMIENSKII, SP. N., A YEAST ASSOCIATED WITH BRINE SHRIMP

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 88, No. 3, p September, 1964 Copyright 1964 Amiierican Society for Microbiology Printed in U.S.A. METSCHNIKOWIA KAMIENSKII, SP. N., A YEAST ASSOCIATED WITH BRINE SHRIMP J. F. T. SPENCER,' H. J. PHAFF, AND N. R. GARDNER' Departnment of Food Science and Technology, University of California, Davis, California ABSTRACT Received for publication 11 April 1964 SPENCER, J. F. T. (University of California, Davis), H. J. PHAFF, AND N. R. GARDNER. MIetschnikowia kamienskii sp. n., a yeast associated with brine shrimp. J. Bacteriol. 88: A description is given of Metschnikowia karnienskii sp. n., four strains of which were isolated from brine shrimp (Artentia salina) collected from a saline lake near Watrous, Saskatchewan, Canada. In addition, we obtained from N. van Uden a fifth strain which was originally isolated by L. J. Wickerham from sporocysts of the trematode Diplostonium flexicauduin parasitizing the hepatopancreas of a lymnaeid snail found in Lake Douglas, Michigan, in which material the yeast was first observed by Kathleen Hussey of Columbia University. All strains were identical and formed long club-shaped asci, containing a single needle-shaped spore, pointed at both ends. M. kanmienskii differs in several of its physiological properties and by the size of its asci from the two previously isolated members of the genus, M. krissii and Ml. zobellii. A key differentiating the three species is given. Metschnikoff (1884) discovered a yeast which was parasitic in Daphnia magna, a fresh-water crustacean. He named it MlIonospora bicuspidata. Only morphological characteristics were reported, because the yeast was not cultured. Kamienski (1899) found a second species associated with the brine shrimp Artemia salina, occurring in a saline body of water near Odessa. Kamienski pointed out that the name Mlonospora was a later homonym of a genus of algae belonging to the Rhodomelaceae, and he changed M1onospora to lietschnikowia. The yeast which grew in the body cavity of Artemia salina, ultimately killing its host, was named Mletschnikowia artemiae. As ' Present address: Prairie Regional Laboratory, Saskatoon, Saskatchewan, Canada. in Jll. bicuspidata, the single needle-shaped spore was pointed at both ends. Kamienski did not culture AI. artemiae; he simply separated it from 31. bicuspidata by the more or less cylindrical shape of the asci in the latter species, as compared with a distinct club-shape in the former. A third species was described by Keilin (1920), who observed it in the body cavity of the dipterous larva Dasehylea obscura, where it acted as a parasite. Keilin proposed the specific name unicuspidata, since the spore was pointed at only one end and rounded at the other. The asci were approximately cylindrical. The organism was not cultured, and thus its physiological properties are not known. It was not until recently when van Uden and Castelo-Branco (1961) isolated two species of the genus in pure culture from marine substrata that they could study for the first time the physiological properties. The two species, M1. zobellii and Ml. krissii, were physiologically distinct. Although in infected daphnias the asci and cells of both yeasts resemble Metschnikoff's drawings of Mll. bicuspidata, a comparison of newly isolated strains with organisms only seen in their respective hosts is impossible by currently accepted procedures in yeast taxonomy. As a result, the name 31. bicuspidata is considered a nomen dubium by van Uden and Castelo-Branco (1961). The generic name Mletschnikowia Kamienski has been challenged a number of times by various authors, but a careful survey of the pertinent literature by van Uden (1962) showed that the name Mletschnikowia was available when Kamienski introduced it in 1899 and it has priority over the frequently used name MIonosporella Keilin (see Lodder and Kreger-van Rij, 1952) or Metschnikowiella Genkel (1913). The present report deals with a third species obtained in pure culture; it was isolated from brine shrimp (A. salina) taken from a saline lake near Watrous, Saskatchewan, Canada. 758

2 NrOL. 88, 1964 METSCHNIKOWIA KAMIENSKII, SP. N. 75'"9 MATERIALS AND METHODS The salt content of the water of the lake near Watrous varies from season to season from 9 to 18% total solids. The major constituents of the salts are chlorides and sulfates of sodium and magnesium. Brine shrimp were taken from the nets as soon as they were landed, and 0.5-g samples were washed with 100 ml of lake water or sterile distilled water. Quantities of 0.2 or 0.5 ml of the wash water were spread on Malt Extract Agar. Colonies were purified by the usual procedures, and the pure cultures were maintained on Malt Agar or Wickerham's (1951) YM maintenance medium. Morphological and physiological properties were determined by the procedures given by Lodder and Kreger-van Rij (1952), except that the number of carbon compounds to study assimilatory reactions was increased as suggested by Wickerham (1951). Carbon assimilation tests were done both by following the growth of colonies on solid medium and by following the growth in liquid medium for 3 weeks. The basal medium for these tests was Yeast Nitrogen Base (Difco), and the carbon sources were present in a concentration of 0.5%. The ph of media with organic acids as the substrate was adjusted to 4.5. Growth at 30 and 37 C was estimated by observing the amount of growth on Malt Extract Agar at these temperatures. RESULTS The new species was represented by four strains from brine shrimp, and a fifth one to be discussed later. Their description is given below. Standard description of 71Ietschnikowia kamienskii sp. n. Growth in malt extract. Growth is rather slow. After 3 days the cells are ovoid to globose, sometimes pear-shaped or slightly elongate, 3.5 to 6.6 by 5.3 to 10,u; occasionally, cells up to 16 are present. Cells reproduce by budding and occur singly, in pairs, or in small clusters or chains. A ring develops gradually. After 1 month, much sediment and a distinct ring are present; pellicle is absent. Growth on malt agar. After 1 month, the streak culture is cream-white, semidull, smooth with very fine transverse striations, and convex. The border is entire with occasional tufts of verv primitive pseudomycelium. Dalmau plates. Aerobically, pseudomycelium is absent or at most a few elongated cells are formed. Anaerobically (under a cover slip), aprimitive pseudomycelium may be formed, consisting of branched chains of ovoid cells, without differentiation between blastospores and pseudomycelial cells. Sporulation. Vegetative cells elongate and become club-shaped, 4 to 7 by 25 to 42 A. Each ascus mother cell develops a single needle-shaped spore, which appears pointed at both ends. The spores are approximately the length of the asci (Fig. 1). Sporulation was observed on yeast extract-malt extract-peptone agar (YM), vegetable agar plus yeast, Gorodkowa agar, brine shrimp agar, and Malt Agar. Sporulation was best in freshly isolated strains and at 10 to 12 C. Fermentation. D-Glucose is fermented slowly; sucrose, D-galactose, lactose, maltose, and raffinose are not fermented. Assimilation of carbon compounds is shown in Table 1. All strains grew slowly. Assimilation of nitrogen compounds: nitrate,- nitrite, -. Growth in vitamin-free medium: absent. Growth on 50% (w/w) glucose-yeast extractagar: absent. Growth at 30 and 37 C: absent. Acid production on glucose-yeast extract-chalkagar: negative. The type culture has been designated as , isolated from Artemia salina collected at Watrous, Little Manitou (saline) Lake, Saskatchewan, Canada. Subcultures have been deposited with the yeast division of the Centraalbureau voor Schimmelcultures, Delft, Holland, and with N. van teden, Department of Microbiology, 13otanical Institute, University of Lisbon, Lisbon, Portugal. Latin diagnosis. Mletschnikowia kamienskii sp. nov. In extracto malti cellulae ovoideae x A, et interdum longovoideae 10-16,(, singulae, binae aut catenatae. Sedimentum et anulus formantur. In agaro malti post unum mensem cultura flavalbida, glabra, subnitida, mollis, margine undulato et piloso. Pseudomycelium primitivum; cellulae in catenis ramosis. Asci clavati, 25-42,u, ascosporae acuiformes, bicuspidatae, ad 1 in asco. Glucosum (lente) fermentatur.

3 760 SPENCER, PHAFF, AND GARDNER J. BACTERIOL. FIG. 1. Asci, ascuis mnother cells, and vegetative cells of MIetschnikowia katmtienskii after I week on Gorodkowa agar at 10 C. The longest ascuis is approximiiately 45, long.

4 I OL. 88, 1964 METSCHNIKOWIA KAMIENSKII, SP. N. 761 Glucosum, galactosum, L-sorbosum, maltosum, saccharum, cellobiosum, trehalosum, D-xylOsum (exigue), ethanolum (var.), D-mannitolum, D- sorbitolum, salicinum, glucono-8-lactonum et Ca-2-ketogluconatum (exigue) assimilantur. Kalium nitricum et natrium nitrosum non assimilantur. Ad crescentiam vitaminae externae necessariae sunt. Isolanta ex Artemiae salinae, Saskatchewanensis, Canada. DiSCUSSION The new species was named in honor of T. Kanmienski, who first observed a representative of the genus Jletschnikowia in brine shrimp and who changed the original generic name from Mklonospora to Metschnikowia. We agree with the arguments presented by van Uden and Castelo-Branco (1961) that a comparison of the presently used combination of physiological and morphological properties of a newly isolated representative of AIetschnikowia with the early descriptions of its morphology in the host does not permit identification of the new isolates with those yeasts only observed in their natural habitat. Although our isolates, represented by four strains, may be the same as M. artemiae Kamienski, the superficial description of the latter species, and the fact that no cultures of Al. artemiae are or ever have been available to permit an amended description, leads us to consider Al. artemiae Kamienski as a nomen dubium. The new species differs from ll. krissii in being able to ferment glucose slowly, by its ability to assimilate D-galactose, L-sorbose, and D-sorbitol, and by its inability to utilize melezitose, glycerol, a-methyl-d-glucoside, and succinate. It differs from ill. zobellii by its inability to assimilate melezitose, a-methyl-d-glucoside, glycerol, adonitol, and succinate. Minor differences between the new species and the two species from the Pacific Ocean are the longer asci (35 to 42,u) of M. kamienskii on sporulation media (such as vegetable agar) as compared with 18 to 24 IA for 31. zobellii and ilm. krissii. However, the last two species also formed very long asci in parasitized Daphnia magna. Another difference is that none of our isolates was able to grow at 30 C, whereas both of van Uden's species had a maximal growth limit of 34 to 35 C. In this connection, it should be mentioned that, in general, sporulation was superior at low temperature, in the range of 6 to 15 C with an optimum near 10 C. At 20 C, very few asci were formed. TABLE 1. Compound Assimilation* Assimilation of carbon compounds Compound Assimilation* D-Glucose... + Glycerol... - D-Galactose... + i-erythritol - L-Sorbose... + Adonitol... - Maltose... + l)ulcitol... - Sucrose... + D-Mannitol. + Cellobiose... + D-Sorbitol. + Trehalose... + a-methyl-d- Lactose... - glucoside Melibiose... - Salicin. + Raffinose... - Glucono-6-lac- Melezitose... - tone. + Inulin Ketoglucon- Soluble starch.. - ate.+ (W) D-Xylose... + (W) 5-Ketoglucon- L-Arabinose... - ate. D-Arabinose - Lactic acid - D-Ribose.. - Succinic acid - L-Rhamnose. - Citric acid. - Ethanol V i-inositol. - * W = weak; V = variable. A key separating the three (cultured) species of.iletschnikowia follows: la. No assimilation of galactose; glucose not fermented-ll. krissii. b. Galactose assimilated, glucose slowly fermented-(2). 2a. Melezitose and a-methyl-d-glucoside assimilated-m1. zobellii. b. Melezitose and a-methyl-d-glucoside not assimilated-ail. kamienskii. When one of the authors (H.J.P.) was visiting Dr. van Uden's laboratory during the summer of 1963, van Uden pointed out that his laboratory possessed a strain very similar to the isolates from brine shrimp. This strain was originally isolated in 1960 by L. J. Wickerham (Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill.) from material collected by Kathleen L. Hussey, School of Public Health, Columbia University, New York, N.Y. Dr. Hussey observed the yeast in the daughter sporocysts (cercaria larval stage) of Diplostomum flexicaudum of the strigeid group of trematodes, which in turn are parasitic in the digestive gland of lymnaeid snails. The samples of Dr. Hussey were collected at Douglas Lake, a fresh water lake in Michigan, and she reported (personal communication to N. van Uden) that very few sporocysts of the hundreds of trematode infections in the snail were parasitized by the yeast. We have

5 762 SPENCER, PHAFF, AND GARDNER J. BACTERIOL. ACKNOWLEDGMENTS We are grateful to N. van Uden for making the culture from trematode larvae available to us. restudied the strain from trematodes, which was made available to use by Dr. van Uden, and found it to be identical to our strains, except that ethanol was utilized strongly by this strain and weakly by our isolates. Kamienski (1899) reported that 111. artemiae was pathogenic to brine shrimp, ultimately killing its host when the body cavity became filled with sporulating asci. We tried to determine the pathogenicity of.1. kamienskii by feeding massive doses of a vegetative culture to brine shrimp, collected in i\iono Lake, California. The shrimp was maintained in natural lake water, which was continually aerated. The yeast cells were consumed by the brine shrimp in about 12 hr, at about the same rate as compressed baker's yeast. In spite of several feedings of Artemia with 3I. kamienskii cells, several days apart, the shrimp remained apparently healthy during the period of observation of 2 to 3 weeks. It is possible that this particular strain of brine shrimp is not susceptible to infection by li. kamienskii, since attempts to cultivate strains of Uetschnikowia from either live or dead shrimp collected at Mono Lake were not successful. Another possibility for the lack of infectivity could be that the high ph of the lake water (approximately 9.6), its salt content (total dissolved solids 51.3 g per liter), and its unusually high fluoride (33 mg per liter) and boron (228 mg per liter) content may have had an adverse effect on the yeast. We also wish to express our appreciation to R. H. Haskins and A. Lutzko for assistance in photographing the cells and asci and to Ellen Barker for technical assistance. Thanks are due to David MIason for supplying the data on AMono Lake water comp)osition and for his hell) in collecting brine shrimp at AMIono Lake. LITERATrURE CITED GENKEL, A Sporovych rastenii, p. 17. Knebel, Moscow. KAMIENSKI, T Notice preliminaire sur la nouvelle espece de Metschnikowia. (Monospora Metschn.) Trav. Soc. Imp. Natural. S. Petersb. 30: KEIIAN, D On a new Saccharomycete Mlonosporiella unicuspidata gen. n. nom., n. sp., parasitic in the body cavity of a dipterous larva (Dasyhelea obscwra Winnertz). Parasitology 12: LODDER, J., AND N. J. W. KREGER-V-AN RIJ The yeasts a taxonomic study. North Holland Publishing Co., Amsterdam. MIETSCHNIKOFF, E Vber eine Sproszpilzkrankheit der Daphnien. Beitrag zur Lehre uber den Kampf der Phagocyten gegen Krankheitserreger. Arch. Pathol. Anat. Physiol. 96: VAN UDEN, N On the nomenclature of the genus Metschnikowia Kamienski. Rev. Biol. 3: VAN UDEN, N., AND R. CASTELO-BRANCO Metschnikowiella zobellii sp. nov. and Al. krissii sp. nov., two yeasts from the Pacific Ocean pathogenic for Daphnia miiagna. J. Gen. Microbiol. 26: WICKERHAM, L. J Taxonomy of yeasts. U.S. Dept. Agr. Tech. Bull. 1029, p