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1 Infection Baltic Saduria entomon (Linnaeus, 1758) (Isopoda, Valvifera) with the Yeast Cryptococcus laurentii (Kufferath) Skinner Author(s): Z. Hryniewiecka-Szyfter, M. T. Smith, A. Kaznowski Reviewed work(s): Source: Crustaceana, Vol. 66, No. 2 (Mar., 1994), pp Published by: BRILL Stable URL: Accessed: 27/02/ :47 Your use the JSTOR archive indicates your acceptance the Terms & Conditions Use, available at. JSTOR is a not-for-prit service that helps scholars, researchers, and students discover, use, and build upon a wide range content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms scholarship. For more information about JSTOR, please contact support@jstor.org. BRILL is collaborating with JSTOR to digitize, preserve and extend access to Crustaceana.

2 Crustaceana 66 (2) 1994,? E. J. Brill, Leiden INFECTION OF BALTIC SADURIA EJVTOMON (LINNAEUS, 1758) (ISOPODA, VALVIFERA) WITH THE YEAST CRTPTOCOCCUS Z^f/??jVT//(KUFFERATH) SKINNER BY Z. HRYNIEWIECKA-SZYFTER Institute Biology, Department Cytology and Histology, A. Mickiewicz PL Poznan, Poland University, Fredry 10, M. T. SMITH Centraalbureau voor Schimmelcultures, Yeast Division, Julianalaan 67, NL-2628 BC Delft, The Netherlands and A. KAZNOWSKI Institute Biology, Department Microbiology, A. Mickiewicz University, Fredry 10, PL Poznan, Poland ABSTRACT Light and electron microscopic and microbiological examination the infected wild Saduria entomon (Linnaeus, 1758) revealed the presence the yeast Cryptococcus laurentii (Kufferath) Skinner, Phagocytosis C. laurentii by granulocytes was observed. This is the first report about the infection isopods with yeasts. R?SUM? La microscopie photonique et?lectronique, et l'examen microbiologique de l'e infect?e de Saduria entomon (Linn?, 1758) ont r?v?l? la pr?sence du champignon Cryptococcus laurentii (Kufferath) Skinner, La phagocytose de C. laurentiipar les granulocytes a?t? observ?e. C'est le premier cas signal? d'une infection d'isopodes par des champignons. INTRODUCTION Some species yeasts are known to be etiological agents crustacean diseases. Van Uden & Castelo-Branco (1961) described Metschnikowiella zobelli and Metschnikowiella krissii, two new yeast species from the Pacific Ocean, pathogenic for Daphnia magna (Straus, 1820). Spencer et al. (1964) isolated four strains Metschnikowia kamienskii from the brine shrimp Artemia salina (Linnaeus, 1758) found in a saline lake in Canada. Metschnikowia bicuspidata (Kamienski, 1899) and Metschnikowia artemiae (Kamienski, 1899) were found in Daphnia magna and Artemia salina from Romania (Codreanu & Codreanu-Balcescu, 1981).

3 206 HRYNIEWIECKA-SZYFTER, SMITH & KAZNOWSKI There is no information available about the infection Crustacea with Cryp tococcus laurentii (Kufferath) Skinner, During light microscopic studies the S. entomon (Linnaeus, 1758) that have been conducted for many years, yeast-like fungi were observed for the first time in some specimens collected in the autumn A microscopic examination the S. entomon collected in the autumn 1990 revealed yeast-like cells in many specimens. Observations S. entomon collected in the spring next year showed yeast cells to be present in the all individuals examined, in some them completely filling the body cavity. The present study reports on light and electron microscopic examinations the wild, yeast-infected specimens S. entomon and the identi fication the yeast isolated from their. MATERIAL AND METHODS The specimens Saduria entomon were collected in the Gulf Gdansk, Baltic sea (54?34'06"N 18?44'09"E) at a depth 50 to 60 m during the spring A group 50 animals was examined two days after collecting. Infected specimens were chosen after yeast cells in the had been ascer tained by light microscopy. The four specimens was acquired by cutting f a walking leg and dripping the fluid on growth media. The yeast cultures were grown and maintained on malt extract and malt extract agar (Van der Walt & Yarrow, 1984) at room temperature for 4 to 5 days. The Dalmau plate technique was used to determine the presence pseu domycelium. The yeast cells were stained using the Gram method and exam ined with light microscopy. The physiological characteristics the four isolates were determined by the conventional techniques applied in yeast classification (Van der Walt & Yarrow, 1984). For tests on carbon growth compounds, cultures were incu bated on a shaker at 28 cycles/min for 28 days at 25? C. Growth on nitrogen compounds was tested on agar plates for 7 days at 25? C. For electron microscopy the was bled from a cut walking leg in to icecold 2.5% glutaraldehyde in the cacodylate buffer, ph 7.2. After 2 hours the was centrifuged at xg for 6 minutes. The pellets were then postfixed in 1% osmium tetroxide for 1 h, washed in the cacodylate buffer, dehydrated a through graded ethanol series, and finally placed in two changes propylene oxide and embedded in Epon. The sections were stained with uranyl acetate and lead citrate and examined in a JEOLCO JEM-7A. RESULTS Infected specimens S. entomon show no visible external morphological symptoms that would distinguish them from unparasitized animals. However,

4 YEAST INFECTION OF SADURIA ENTOMON 207 there are differences in the microscopic picture the in particu lar infected specimens under study which are shown in figs. 1 and 2. Fig. 1 shows in which budding cells yeasts can be discerned as well as fairly numerous haemocytes, mostly swollen because containing phagocytosed yeast cells. The these specimens was transparent and clear. The presented in fig. 2 was opaque-white, and the specimens that had it moved slower and usually died after two weeks. No haemocytes can be observed in this, and it was filled with non-budding ovoidal yeast cells. The microbiological examination the opaque-white the four specimens showed the presence yeast cells after 3 to 5 days cultivation on malt agar at room temperature. Under light microscopy the cells were Gram positive, single, ovoidal to elongated. After 7 to 14 days the culture on malt agar was cream-yellowish-pinkish-orange. The surface the culture was smooth and glossy. Pseudomycelium was absent by the Dalmau plate technique. The results the growth on C- and N-compounds are listed in table I. Visible C02 production from glucose was not observed. On the basis the morphological and physiological data and using the computer 'Yeast Identi fication: PC Program' Barnett et al. (1990), the four isolates were identified as Cryptococcus laurentii (Kufferath) Skinner, Electron micrography the yeast cells C. laurentii from show the presence a thin electron dense outer layer and a thick electron lucent inner layer (figs. 3, 4). Fig. 5 shows that the wall the yeast cell is multi layered. The cytoplasm the cells C. laurentii is electron dense, and therefore Figs. 1, 2. Saduria entomon (L., 1758), phase contrast micrographs showing fresh two variously infected specimens. 1, clear containing budding yeast cells (y) and swollen haemocytes (H) with phagocytosed yeast cells, X 360; 2, opaque white filled with non budding yeast cells, x 190.

5 208 HRYNIEWIEGKA-SZYFTER, SMITH & KAZNOWSKI Table I Physiological characteristics four isolates Cryptococcus laurentii isolated from Saduria entomon Fermentation absent Growth D-glucose D-galactose L-sorbose V D-glucosamine D-ribose D-xylose L-arabinose D-arabinose L-rhamnose Sucrose Maltose oe-oc-trehalose cc-methyl-d-glucoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose Melezitose Inulin Soluble Glycerol Erythritol Ribitol Xylitol starch L-arabinitol D-glucitol D-mannitol Galactitol Myo-inositol D-glucoco-?-lactone D-gluconate DL-lactate V Succinate Citrate Methanol Ethanol - Nitrate Nitrite Ethylamine V L-lysine Cadaverine - Creatine - Creatinine Vitamin-free 25? C 37? C medium such cell organd?es as mitochondria, ribosomes or the endoplasmic reticulum are indistinguishable here. In a non-budding yeast cell the nucleus is in the centre (fig. 3). At the poles clusters electron lucent spherules can be found (fig. 3). In a budding yeast cell electron lucent spherules gather only at one pole, while in the opposite part a characteristic structure can be observed (fig. 4). It resembles the 'voluminous spongy reticulate body' described in a young vegeta tive eel the yeast Metschnikowia artemiae (Codreanu & Codreanu-Balcescu, 1981). In the some specimens yeast cells could be observed which have lost their cell walls, presenting protoplasts (fig. 6). Their ultrastructure differs from that walled cells the yeast. The protoplasts are irregular, oval or amoeboid. Their cytoplasm is packed with ribosomes and contains a rough endoplasmic reticulum and elongated mitochondria (fig. 6). The protoplasts do not contain the electron lucent spherules present in walled cells. Electron microscopy corroborated the light microscopic observation that the haemocytes S. entomon phagocytose the cells C. laurentii. There are three types haemocytes in the S. entomon: hyaline cells, intermedi ate cells, and granulocytes (Hryniewiecka-Szyfter, 1986). In the specimens S. entomon infected with C. laurentii, granulocytes are the primary phagocytic haemocytes. Fig. 7 shows a granulocyte containing eight phagocytosed cells

6 YEAST INFECTION OF SADURIA ENTOMON 209 Figs Cryptococcus laurentii (Kufferath) Skinner. 3, electron micrograph non-budding cells from ; N, nucleus; S, lucent x spherules; , electron micrograph a budding cell from ; S, lucent spherules; r, reticulate x body; , electron micrograph a cell a showing multilayered wall, X , electron micrograph a protoplast a cell packed with ribosomes, containing a rough endoplasmic reticulum and an elongated mitochondrium (M), x , electron micrograph a granulocyte with 8 phagocytosed cells, X , electron micrograph a hyaline haemocyte without phagocytosed cells, X 4920.

7 210 HRYNIEWIECKA-SZYFTER, SMITH & KAZNOWSKI C. laurentii. In the this same not specimen hyaline haemocytes changed and do not contain phagocytosed yeasts (fig. 8). are DISCUSSION Numerous studies have shown that the fungal diseases have become one the most important types diseases cultured and wild aquatic crustacean populations (review articles: Unestam, 1973; Johanson, 1983). This is the first report the yeast infection the an aquatic isopod. The species we have identified, Crytococcus laurentii (Kufferath) Skinner, is a microorganism infecting the S. entomon in which it multiplies rapidly by budding. In some infected specimens the cells C laurentii fill all the haemocoelic space. Their is opaque-white and they show increased mortality. The wall-free protoplasts C laurentii described in the present paper may appear as a result the action lytic enzymes in the S. ento mon. A review studies on the emergence protoplasts from yeasts by enzymatic digestion is given in Peberdy (1979). The haemocytes Crustacea play an important role in the defense reaction against pathogenic microorganisms through phagocytosis, encapsulation and nodule formation (review article: Bauchau, 1981). The results this study indicate that phagocytosis in S. entomon is the main, if not only, response to infection with C. laurentii. As mentioned in the Introduction, in the microscopic examinations the a group wild specimens S. entomon collected in 1989 a few infected animals were found, while all the wild animals (out a group 50 under study) collected in 1991 had yeast cells in their. Thus, it may be presumed that there is an epizootic spreading among the Baltic popula tion S. entomon under study. REFERENCES Barnett, J. A., R. W. Payne & D. Yarrow, Yeast Identification: PC Program, version 2. (J. A. Barnett, Norwich). Baughau, A. G., Crustaceans. In: N. A. Ratcliffe & A. F. Rowley (eds), Invertebrate blood cells: (Academic Press, New York). Codreanu, R. & D. Codreanu-Balgescu, On two Metschnikowia yeast species producing hemocoelic infection in Daphnia magna and Artemia salina (Crustacea, Phyllopoda) from Romania. Journ. Invertebr. Pathol., 37: Hryniewiegka-Szyfter, Z., Light and electron microscopic studies haemocytes Mesidotea entomon L. (Crustacea, Isopoda). Bull. Soc. Amis Sei. Lett. Poznan, (D) 25: Johnson, P. T., Diseases caused by viruses, rickettsiae, bacteria, and fungi. In: A. J. Provenzano (ed.), The biology Crustacea, 6: (Academic Press, New York). Peberdy, J. F., Fungal protoplasts: isolation and fusion. Ann. Rev. Microbiol., 33: Spencer, J. F. T., H. J. Phaff & N. R. Gardner, Metschnikowia kamienskii sp. n., a yeast associated with brine shrimp. Journ. Bacteriol., 88: Unestam, T., Fungal diseases Crustacea. Rev. med. Vet. mycol., 8: 1-20.

8 YEAST INFECTION OF SADURIA ENTOMON Van Uden, N. & R. Castelo-Branco, Metschnikowiella zobellii sp. nov. and M. krissii sp. nov., two yeasts from the Pacific Ocean pathogenic for Daphnia magna. Journ. Gen. Microbiol., 26: Van der Walt, J. P. & D. Yarrow, Methods for isolation, maintenance, classification and identification yeast. In: N. J. W. Kreger-Van Rij (ed.), The yeasts, a taxonomic study: (Elsevier Science Publishers, Amsterdam). Received for publication 9 November 1992.