Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the ‘naked’ flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.

Recently, large discrepancies have been identified between microsporidian systematics based on molecular and traditional characteristics. In the current study the 530f–580r region of the rRNA gene of eight microsporidian species was cloned and sequenced. Included were two unclassified species of Microsporidium Balbiani, 1884 and an unidentified microsporidian that infects the musculature of different sea bream species. Sequence identities in excess of 98% indicated that these three species almost certainly are members of the same genus. Phylogenetic analyses of all microsporidian sequence data available for this region of the gene (20 species) and for partial small subunit sequences (51 species of 21 genera) revealed these species to be distinct from the family Pleistophoridae Doflein, 1901 and closely related them to the genus Spraguea Weissenberg, 1976. This clade was found to comprise a sister taxon to that containing the vast majority of fish-infecting species. Broad cladistic divisions were found between terrestrial insect-infecting and fish-infecting species, which together are distant from the aquatic insect-infecting microsporidia. The rRNA gene of certain fish-infecting genera was found to be more highly conserved than previously reported. This has implications for its utility in diagnostic assays and phylogenetic studies at, or close to, the species level.

Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid × diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid × diploid] conjugants with mating pairs involving an aneuploid partner [diploid × “star”], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the “abort” signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the “terminal commitment point”, a developmental stage in normal [diploid × diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.

Psilotricha acuminata was described by Stein in 1859 as the type species of the ciliate genus Psilotricha Stein, 1859. The ciliate has rarely been found since, and its infraciliature has never been described with the aid of silver-impregnation techniques. We have found P. acuminata Stein, 1859 in soil samples from upland grassland in Scotland (U.K.). Living and healthy organisms of P. acuminata are oblong in outline, and dorso-ventrally compressed. They closely resemble ciliates of the genus Euplotes. The main morphological features used for identification of P. acuminata are the very long and sparse cirri, and the two macronuclei. When the ciliate crawls, the cirri appear stiff and directed backwards. Specimens observed from the ventral side have a protruding anterior end, a rounded or acuminate posterior pole, and a “beak-like” projection to the left side of the posterior end. The ciliate shares characters with the Euplotidae (body shape and reduced ciliature) and with the Oxytrichidae (marginal rows, macronuclei, reduced number of transverse cirri). Because the arrangement of the silver-impregnated infraciliature was unknown, and as the only description of the ciliate was that of Stein (1859a, b), the genus Psilotricha became confused with other hypotrich genera, especially Urospinula Corliss, 1960. Here we provide a full redescription of P. acuminata based on living and silver-impregnated specimens, and a revision of the genera Psilotricha Stein, 1859 and Urospinula Corliss, 1960. We resurrect the genus Urospinula, and give an emended diagnosis for both genera. The species now included within the genus Psilotricha are P. acuminata Stein, 1859 (type species); Psilotricha viridis (Penard, 1922) Kahl, 1932; and Psilotricha geleii (Gelei, 1954) Stiller, 1974. Psilotricha viridis sensu Kahl, 1932 and Psilotricha dragescoi Grolière, 1975 are considered incertae sedis.

Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small-subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal-fungal divergence, together with the choanoflagellate-Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal-fungal divergence.

We investigated growth rates, grazing rates, and prey selection of Polykrikos kofoidii when feeding on several species of red-tide and/or toxic dinoflagellates. Polykrikos kofoidii ingested all prey species used in this study, exhibiting positive growth on Lingulodinium polyedrum, Scrippsiella trochoidea, Ceratium furca, Gymnodinium catenatum, Gyrodinium impudicum, Prorocentrum micans, and the toxic dinoflagellate Amphidinium carterae, but not on P. minimum. Specific growth rates of P. kofoidii increased rapidly with increasing density of L. polyedrum, S. trochoidea, C. furca, and G. catenatum before saturating between 500–2,000 ng C ml⁻¹. Specific growth rates increased continuously when P. kofoidii was fed the other prey species. Maximum specific growth rates of P. kofoidii on G. catenatum (1.12 d⁻¹), S. trochoidea (0.97 d⁻¹), and L. polyedrum (0.83 d⁻¹) were higher than those on C. furca (0.35 d⁻¹), A. carterae (0.10 d⁻¹), P. micans (0.06 d⁻¹), G. impudicum (0.06 d⁻¹), and P. minimum (−0.03 d⁻¹). Threshold prey concentrations (where net growth = 0) were 54–288 ng C ml⁻¹. Maximum ingestion and clearance rates of P. kofoidii on these dinoflagellates were 5–24 ng C pseudocolony⁻¹ d⁻¹ and 1.0–5.9 μl pseudocolony⁻¹ h⁻¹, respectively. Polykrikos kofoidii strongly selected L. polyedrum over S. trochoidea in prey mixtures. Polykrikos kofoidii exhibited higher maximum growth, ingestion, and clearance rates than previously reported for the mixotrophic dinoflagellate Fragilidium cf. mexicanum or the heterotrophic dinoflagellates Protoperidinium cf. divergens and P. crassipes, when grown on the same prey species. Grazing coefficients calculated by combining field data on abundances of Polykrikos spp. and co-occurring red-tide dinoflagellate prey with laboratory data on ingestion rates obtained in the present study suggest that Polykrikos spp. sometimes have a considerable grazing impact on prey populations.

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20:1(n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2Δ7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.

A new genus and species combination are proposed for Urocryptum tortum n. gen., n. comb., a scuticociliate with a polymorphic life cycle. This marine ciliate was isolated from a sample taken at Gokasho Bay in Mie Prefecture (Japan). Specimens from different phases of the growth cycle were examined in vivo and with two silver staining techniques. Three life-history stages were observed: an exponential growth phase stage (trophont), a stationary phase stage (tomite), and finally a resting stage (cyst). The exponential growth form is laterally flattened and ovoid; it has 20–24 somatic kineties (SK) and a typical complement of scuticociliate oral structures. Polykinetid 1 (Pk1) has two longitudinal files of 6 kinetosomes (Ks); sometimes one or two additional kinetosomes are located anteriorly. Polykinetid 2 (Pk2) has two files of 6 or 7 Ks; a third file of three widely spaced kinetosomes is located on its right side, as well as a small curved row of 5 Ks positioned on the right hand side of the posterior end of Pk2. Polykinetid 3 (Pk3) has 3 rows of 4, 5, and 7 Ks's perpendicular to the haplokinety (Hk) or paroral membrane. The stationary growth phase cell is spindle-shaped and has a similar number of SK that are much more closely spaced than in exponentially growing specimens. Oral infraciliary structures are reduced in size, having fewer kinetosomes and being positioned more anteriorly in the cell. Pk1 is composed of two files of 5–6 Ks, Pk2 has only two files of 6–7 Ks, and Pk3 has two to three rows of 3–4 Ks. The Hk is displaced anteriorly and becomes straight, losing the shape typical of exponential growth phase cells. Cysts are pyriform with a wide cyst wall; no infraciliary structures were visible.

Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marinus, a pathogen of the eastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell culture was propagated from infected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts, were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles with vacuoplasts. There were micropores on the inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles, and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggesting a function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and may specialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infected oyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose, hypha-like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangial stages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.

Large miliolid foraminifers bear various types of algal endosymbionts including chlorophytes, dinoflagellates, rhodophytes, and diatoms. Symbiosis plays a key role in the adaptation of large foraminifera to survival and growth in oligotrophic seas. The identity and diversity of foraminiferal symbionts, however, remain largely unknown. In the present work we use ribosomal DNA (rDNA) sequences to identify chlorophyte endosymbionts in large miliolid foraminifera of the superfamily Soritacea. Partial 18S and complete Internal Transcribed Spacer (ITS) rDNA sequences were obtained from symbionts of eight species representing all genera of extant chlorophyte-bearing Soritacea. Phylogenetic analysis of the sequences confirms the previous fine structure-based identification of these endosymbionts as belonging to the genus Chlamydomonas. All foraminiferal symbionts form a monophyletic group closely related to Chlamydomonas noctigama. The group is composed of seven types identified in this study, including one previously morphologically described species, Chlamydomonas hedleyi. Each of these types can be considered as a separate species, based on the comparison of genetic differences observed between other established Chlamydomonas species. Several foraminiferal species share the same symbiont type, but only one species, Archaias angulatus, was found to bear more than one type.

Large miliolid foraminifers of the subfamily Soritinae bear symbiotic dinoflagellates morphologically similar to the species of the “Symbiodinium” complex, commonly found in corals and other marine invertebrates. Soritid foraminifers are abundant in coral reefs and it has been proposed that they share their symbionts with other dinoflagellate-bearing reef dwellers. In order to test this hypothesis, we have analysed partial large subunit ribosomal DNA sequences from dinoflagellates symbionts obtained from 28 foraminiferal specimens, and compared them to the corresponding sequences of Symbiodinium-like endosymbionts from various groups of invertebrates. Phylogenetic analysis of our data shows that all soritid symbionts belong to the “Symbiodinium” species complex, within which they form seven different molecular types (Fr1–Fr7). Only one of these types (Fr1) branches within a group of invertebrate symbionts, previously described as type C. The remaining six types form sister groups to coral symbionts previously designed as types B, C, and D. Our data indicate a high genetic diversity and specificity of Symbiodinium-like symbionts in soritids. Except for type C, we have found no evidence for the transmission of symbionts between foraminifers and other symbiont-bearing invertebrates from the same localities. However, exchanges must have occurred frequently between the different species of Soritinae, as suggested by the lack of host specificity and some biogeographical patterns observed in symbiont distribution. Our data suggest that members of the subfamily Soritinae acquired their symbionts at least three times during their history, each acquisition being followed by a rapid diversification and independent radiation of symbionts within the foraminiferal hosts.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051–1.059/g/ml in a microgradient procedure, and 1.102–1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [³H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [¹⁴C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-α-difluoromethylornithine (DFMO) but not by DL-α-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N¹-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs, which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.