Protistology 4 (3), 227244 (2006) Protistology Structure and development of Pelomyxa gruberi sp. n. (Peloflagellatea, Pelobiontida) Alexander O. Frolov 1, Andrew V. Goodkov 2, Ludmila V. Chystjakova 3 and Sergei O. Skarlato 2 1 Zoological Institute RAS, St. Petersburg, Russia 2 Institute of Cytology RAS, St. Petersburg, Russia 3 Biological Research Institute of St. Petersburg State University, Russia Summary The general morphology, ultrastructure, and development of a new pelobiont protist, Pelomyxa gruberi, have been described. The entire life cycle of this eukaryotic microbe involves an alteration of uni and multinucleate stages and is commonly completed within a year. Reproduction occurs by plasmotomy of multinucleate amoebae: they form division rosettes or divide unequally. Various surface parts of this slowlymoving organism characteristically form fingershaped hyaline protrusions. Besides, during the directed monopodial movement, a broad zone of hyaline cytoplasm with slender fingershaped hyaline protrusions is formed at the anterior part of the cell. In multinucleate stages up to 16 or even 32 nuclei of a vesicular type may be counted. Individuals with the highest numbers of nuclei were reported from the southernmost part of the investigated area: the NorthWest Russia. Each nucleus of all life cycle stages is surrounded with microtubules. The structure of the flagellar apparatus differs in individuals of different age. Small uninucleate forms have considerably fewer flagella per cell than do larger or multinucleate amoebae but these may have aflagellated basal bodies submerged into the cytoplasm. In young individuals, undulipodia, where available, emerge from a characteristic flagellar pocket or tunnel. The basal bodies and associated rootlet microtubular derivatives (one radial and one basal) are organized similarly at all life cycle stages. There is a thinwalled cylinder in the flagellar transition zone, and an electrondense column above that zone. In the separate nonmotile undulipodia the arrangement of axoneme microtubules deviates from the typical 9+2 eukaryotic pattern. In the cytoplasm of P. gruberi two types of rodshaped endocytobionts are present: (1) large bacteria with a pronounced longitudinal cleft, and (2) smaller methanogenlike bacteria. Key words: systematics, life cycle, Pelobiontida, Pelomyxa gruberi sp. n., ultrastructure, cytoskeleton © 2006 by Russia, Protistology 228 · Alexander O. Frolov et al. Introduction almost stagnant permanent water bodies. The samples were taken near the bank, at a depth of 570 cm. The systematics of the genus Pelomyxa Greeff 1874 Attempts to establish Pelomyxa cultures failed, but the was influenced for a long time by two opposite views collected amoebae survived for up to 14 months in with regard to both qualitative and quantitative samples put in hermetically sealed 300 ml vessels and composition of this taxon. On the one hand, nearly 20 kept at about 10°С. Alive individuals were investigated species had been described within a century resulting in closed microaquaria, with a volume of 2.5 ml3, from numerous observations of Pelomyxalike amoebae connected via a running system with a 0.5 l vessel filled (Gruber, 1884; Penard, 1902; Goodkov et al., 2004). with water and silt from their natural habitat. The On the other hand, the validity of most of these species system was maintained at 10°С and transferred to room was often doubted (Page, 1981, 1988; Whatley and temperature only for observation. ChapmanAndresen, 1990). Results of studies into the Leika microscope equipped with visualisation life cycle of P. palustris (the type species) seemed to systems on the basis of Panasonic 650 CCTV + Canon resolve the question in favour of monotypy of the genus EOS350D and PC P4 was used. All measurements were Pelomyxa (ChapmanAndresen, 1978, 1982). It was made on live individuals, with the help of the image postulated that all Pelomyxa species ever described were analysis system IT v.2.2 (UTHSCSA). merely particular stages of the complex life cycle of the For electron microscopy the amoebae were fixed sole polymorphic species P. palustris (Whatley and with a cocktail of 5% glutaraldehyde and 0.5% OsO4 ChapmanAndresen, 1990). Since then no other (1:1) on 0.1 M cacodylate buffer. Fixation was detailed studies of multinucleate pelobionts followed, performed on melting ice in the dark for 4 h, followed and until recently the above concept remained by the fixative replacement in 15 min. Then fixed dominating in special protistological literature (Whatley amoebae were washed in 0.1 M cacodylate buffer (15 and ChapmanAndresen, 1990; Page and Siemensma, min) and postfixed with 2% OsO4 on 0.1 M cacodylate 1991; Brugerolle, 1991; Brugerolle and Patterson, 2002; buffer in total darkness on melting ice (1 h). Goodkov et al., 2004). After a transition through a graded ascending The situation has dramatically changed when alcohol series, the material was embedded in Epon Frolov et al. (2005a, 2005b) reported results of their Araldite mixture. To facilitate the preparation of ultrastructural studies of such "life cycle stages" of P. ultrathin sections, the objects embedded in the resin palustris as P. binucleata (Gruber 1884) ("division were treated with 10% solution of hydrofluoric acid product of large, grey type of P. palustris" sensu Whatley (HF). Ultrathin sections were cut with a Reichert and ChapmanAndresen, 1990) and P. prima (Gruber ultratome and viewed in the Tesla BS300 electron 1884) ("young growth phase of P. palustris" sensu microscope. Whatley and ChapmanAndresen, 1990). The obtained morphological characters allowed reliable differen Results tiation between P. binucleata, P. prima, and the type species P. palustris. The most considerable differences Pelomyxa gruberi sp. n. (Figs 110). involved basal parts of flagella, nuclear organization and Diagnosis: Rounded (feeding stages) or elongated cell surface of examined species of Pelomyxa. The cylindrical (locomotive stages) amoeboid organisms. A provided evidence (Frolov et al., 2004, 2005a, 2005b) welldeveloped broad zone of hyaline cytoplasm with resumed an interest to the previous question about the slender fingerlike hyaline protrusions formed at the true biodiversity of multinucleate pelobionts. anterior body end during locomotion. Cell diameter In the present paper, a new species of pelobionts, varies from 80 !m (young uninucleate individuals) to Pelomyxa gruberi, is described. The species name is 350 !m (multinucleate locomotive forms). In the life given in honour of Professor August Gruber (University cycle, uninucleate developmental stages alternate with of Freiburg, Germany), whose detailed descriptions of multinucleate ones. Reproduction occurs by plasma Pelomyxalike amoebae (Gruber, 1884) anticipated tomy of multinucleate amoebae: they form division modern studies of pelomyxoid diversity. rosettes or divide unequally. The cytoplasm clearly differentiated into ectoplasm and endoplasm. Vesicular Material and Methods nuclei (132 per cell) with a single central nucleolus. Nuclear envelope surrounded with microtubules in all Pelomyxa amoebae were collected in freshwater life cycle stages. Flagella are numerous, nonmotile; basins in the NorthWest Russia (Sosnovo Village, the axoneme with a nonstandard microtubular pattern. Leningrad region, 60° 30' N, 30° 30' E and Lyady Long basal bodies deeply submerged into the cytoplasm Village, the Pskov Region, about 58° 35' N, 28° 55' E). and associated with microtubular derivatives of two The amoebae were found in silt samples from fully or types. First type derivatives (over 150 radial micro Protistology · 229 tubules) start from the lateral basal body surface. Second After numerous observations within the latest three type derivatives (about 60 microtubules) are represented years, we established in the life cycle of P. gruberi the by one or two compact bundles originating from the alternation of uninucleate and multinucleate stages bottom of the basal body. A thinwalled cylinder is (Fig. 1). Prevalence of uninucleate individuals and mass present in the transition zone, an electrondense appearance of multinucleate stages in P. gruberi column is located above it. In the cytoplasm two types micropopulations occurred approximately once a year. of rodshaped endocytobionts are present: (1) large Interestingly, this cyclic recurrence was not season bacteria with a pronounced longitudinal cleft, and (2) related. Indeed, five micropopulations of P. gruberi were smaller methanogenlike bacteria. observed weekly in the Osinovskoye Lake from October Amoebae feed on small detritus particles, mostly to December, 2004. The five examined habitats were of vegetative origin. No glycogen bodies are formed in small silted anthropogenic bays on the west shore of the any life cycle stages. Cytoplasm colour varies from light lake. The bays, located 3050 m apart, had similar depth beige to dark brown. and bottom relief, in addition to identical shore Type locality: Sosnovo Village, the Leningrad landscapes. Table 1 summarises data on the proportion Region, 60° 30' N, 30° 30' E, NorthWest Russia. of uninucleate and multinucleate forms in these Type material: Holotype, slide N 934; Paratypes, micropopulations. Multinucleate amoebae were slides N 935 and N 936; deposited in the Type Slide registered only in one of the five above habitats (bay 4). Collection, Laboratory of Unicellular Organisms, Subsequent observations showed that in bays 13 and 5 Institute of Cytology RAS, St. Petersburg, Russia. the multinucleate