The taxonomic position and phylogenetic relationships of the Pelobionta, an amitochondriate amoeboflagellate group, are not yet completely settled. To provide more information, we obtained sequences for the large subunit rDNA gene, the gene for translation elongation factor 1α, and for a large part of the gene encoding translation elongation factor 2 from a representative of this group, Mastigamoeba balamuthi (formerly Phreatamoeba balamuthi). The gene for the large subunit rDNA was unusually large compared to those of other protists, a phenomenon that had previously been observed for the gene encoding the small subunit rDNA. Phylogenetic reconstruction using a maximum likelihood method was performed with these sequences, as well as the gene encoding the small subunit rDNA. When evaluated individually, the M. balamuthi genes for the small and large subunit rDNAs and elongation factor 1α had a most recent common ancestor with either the Mycetozoa (slime molds) or with Entamoeba histolytica. A clade formed by M. balamuthi, E. histolytica, and Mycetozoa was not rejected statistically for any of the sequences. A combined maximum likelihood analysis using 3,935 positions from all molecules suggested that these three taxonomic units form a robust clade. We were unable to resolve the closest group to this clade using the combined analysis. These findings support the notion, which had previously been proposed primarily on cytological evidence, that both M. balamuthi and E. histolytica are closely related to the Mycetozoa and that these three together represent a major eukaryotic lineage.

The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular-genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflagellates. To test for evidence of serological epitopes common to P. marinus and dinoflagellates, we probed 19 free-living and 8 parasitic dinoflagellate, or dinoflagellate-like, species for cross-reactivity with polyclonal antibodies to P. marinus. Three of 19 free-living dinoflagellates (16%), and 7 of 8 parasitic dinoflagellates (88%) were labeled by anti-P. marinus antibodies. In reciprocal immunoassays using polyclonal antibodies to the Hematodinium sp. dinoflagellate parasite of Norway lobsters, Nephrops norvegicus, P. marinus and the same 7 parasitic dinoflagellates labeled by anti-P. marinus antibodies, were again labeled. The dinoflagellate-like parasite of prawns Pandalus platyceros was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody-binding epitopes on cells of P. marinus and several dinoflagellates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflagellates suggests a closer phylogenetic link to the syndinean dinoflagellate lineage. The consistent failure of the dinoflagellate-like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Hematodinium-species parasitic dinoflagellates.

Soil samples (varying in granularity) from four natural sites were cultured in microcosms to determine small-scale patchiness in abundance and diversity of gymnamoebae. Eighty grams of the same thoroughly mixed soil, either moistened with distilled water (− nutrients) or supplemented with an equivalent vol. of organically enriched water ( nutrients), were placed in covered glass jars and incubated for 14 d (25 °C). Abundances (number/gram soil) were assessed in each of 3 core samples (5–10 mm apart). Assay precision was estimated to be ± 4%. Abundances were similar in the 3 closely-spaced samples, but occasional samples had higher abundances, probably representing localized enriched sites (“nutrient hot spots”). Diversity within the triplicate, closely spaced samples varied substantially. Mean abundance and diversity of amoebae were consistently higher in organically enriched soil and in soil of increasing granularity. Field samples collected directly from two of the sites showed similar patterns of abundance and diversity as found in the experimental studies, indicating substantial small-scale compartmentalization of soil protist communities. These data provide evidence of soil eukaryotic microbiocoenoses and indicate that soil microfauna may encounter wide variations in resources and prey communities as they migrate within small distances of several millimeters or less.

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to −125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.

Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non-photosynthetic dinoflagellate, Crypthecodinium cohnii: actin, beta-tubulin, hsp70, BiP, hsp90, and mitochondrial hsp10. Beta-tubulin, hsp70, BiP, and hsp90 were found to be useful for intra-alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa-dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.

One liter of rumen fluid containing 4.7 × 10⁴ ciliates/ml, representing four genera including nine species of ciliates from a Japanese sika deer was inoculated into two unfaunated Japanese shorthorn calves. Two weeks after inoculation, all species originally present in the inoculum were subsequently detected in the rumen fluid of one or both calves. Ciliate densities ranged from 10⁵–10⁶ cells/ml over the remainder of the 33-wk experiment. The inoculum contained Diplodinium rangiferi. which lacks caudal appendages, as is characteristic for the species. However, three weeks later, the rumen fluid of both calves contained D. rangiferi, which possesses caudal appendages varying from a single spine to multiple spines with a complicated furcate appearance. The caudal spines of D. rangiferi did not disappear during the experiment, even when the diet of the calves was switched to the ration of sika deer from which the inoculum was obtained.

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic ‘B’ subunit of vacuolar ATPase of B. hominis (HE87–1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87–1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.

The physiological effects on isotropically heated populations of Oxytricha bifaria cultured at 24 °C were investigated. At 34.6 °C ciliates became inert, and did not adaptively react to either cold or warm microgradients; they neither moved towards the favorable cold thermal source nor escaped from the unfavorable warm one. The inert oxytrichas were only able to perform the Side-Stepping Reaction (SSR) on the same spot. However, mobile ciliates at 31.6 °C reacted to the cold microgradient by immediately orienting themselves towards its source, without accelerating but reducing their SSR frequency. Moreover, in a warm microgradient such ciliates immediately increased their SSR frequency, then moved away from the thermal source. At 34.6 °C the behavior of ciliates was not-adaptive—not acting to guide the organisms to more favorable conditions—whereas at 31.6 °C it was still clearly adaptive. Therefore, the locomotory inertness of the oxytrichas at 34.6 °C was the result of thermal stress rather than their behavioral response to the environmental isotropy, in contrast to populations of the same species made inert at 9 °C.

Ceratium furca is a primarily photosynthetic dinoflagellate also capable of ingesting other protists. During 1995 and 1996, we documented the abundance of C. furca in Chesapeake Bay and determined grazing rates on prey labeled with fluorescent microspheres. Abundance usually remained below 20 cells ml⁻¹, although the species was capable of localized late-summer blooms (≤ 478 cells ml⁻¹) in the more saline lower to mid-Bay region. Feeding rates ranged from 0 to 0.11 prey dinoflagellate⁻¹ h⁻¹ or from 0 to 37 pg C dinoflagellate⁻¹ h⁻¹ and were highest at lower salinities. Clearance rates averaged 2.5 ± 0.35 μl dinoflagellate⁻¹ h⁻¹. Impact of C. furca feeding on prey populations was higher in the lower Bay, averaging 67% of Strobilidium spp. removed d⁻¹. Ingestion rates were positively correlated with prey abundance and dissolved inorganic nitrogen, but negatively with salinity, depth, dissolved inorganic phosphorus, and inorganic P:N ratio. Daily consumption of prey biomass by C. furca averaged 4.6% of body carbon, 6.5% of body nitrogen, and 4.0% of body phosphorus, with maximal values of 36, 51, and 32%, respectively. Thus, the ability to exploit an organic nutrient source when inorganic nutrients are limiting may give C. furca a competitive advantage over purely photosynthetic species.

Microtubule dynamics in Paramecium caudatum were investigated with an anti-α-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-α-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 μmol min⁻¹mg⁻¹. Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx_2–4-Cys-Xxx_5–7 -Cys.