Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

Cyst-forming amoebae of the genus Korotnevella (Amoebozoa: Dactylopodida), with description of two new species.

The genus KorotnevellaGoodkov, 1988 (Amoebozoa: Dactylopodida) comprises amoebae with a complex cell coat that include a layer of scales covering the entire cell surface. Until now no Korotnevella species were known to form cysts. Here we describe two new species of genus Korotnevella and provide a light- and electron-microscopic report of encystment in three species (K. stella (Schaeffer, 1926) Goodkov, 1988, K. limbata n. sp. and K. heteracantha n. sp.) of this genus. Korotnevella limbata has two peculiar wide rims along the upper margin of basket scales forming two short spines at their junctions. The basket scales of K. heteracantha are very similar to those of K. bulla, but have a different spine length and a closed latticework basket with polygonal elements. All three studied species have similar cyst wall consisting of three layers. The outer layer contains scales of trophic stage; the middle one includes so called "cyst scales" with meshwork walls; and the inner one is composed of fibrillar material. Cyst structure of this sort appears to be unique for lobose amoebae and probably represents an autapomorphy for genus Korotnevella.

Balamuthia mandrillaris: the multiple nuclei of Balamuthia amebas; their location, activity, and site of development.

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.