Cyst formation in a freshwater strain of the colonial freshwater choanoflagellate Desmarella moniliformis Kent (Protozoa: Choanoflagellida) has been studied with light and electron microscopy for the first time. Batch cultures inoculated with motile vegetative cells start to produce cysts within 3 days during the exponential phase of growth. Cyst production proceeds until in late stationary phase there is a preponderance of cysts. Transfer of cysts to fresh medium results in limited excystment. Encystment involves the production of electron-dense fibrillar wall material, firstly around the neck of the cell and then around the posterior end. As the wall material is deposited the neck of the cell elongates and the dictyosome rotates from the horizontal to vertical plane. The number of mitochondrial profiles seen in individual sections of cells increases. Finally the neck of the cell is retracted, the flagellum and collar tentacles are withdrawn, and the bottom of the neck of the cyst wall is sealed with a diaphragm of wall material. Excystment, which has not been observed directly, appears to involve the disruption of the wall at the base of the neck, the remainder of the cyst wall remains intact. Comparisons are made between encystment in Desmarella and cyst development in other protists.

Two isoforms of α-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.

Isolates of Sterkiella (Oxytrichidae, Stichotrichia, Ciliata) are commonly used to study macronuclear development. These organisms respond to changes in food abundance variably by encystment-excystment, conjugation, cannibalism or rescaling cell size. An isolate of Sterkiella histriomuscorum (previously Oxytricha fallax and O. trifallax) is used because two complementary mating types are available. We provide observations on conjugation in cultures of this isolate. Using synchronous samples of conjugants, the timing of stages of nuclear divisions during conjugation was determined. Following ex-conjugant cultures over time, the onset of clonal aging and senescence is described. Cells become sexually mature after a brief period of “adolescence”, during which time selfing is possible. Senescent cultures are less vigorous, unable to conjugate and encyst more readily. Excystment survival decreases with clonal age. These results can serve as reference for long-term cultures of this species and for analysing particular stages of developmental processes during conjugation.

The sensitivity to ultraviolet radiation (UVR, 280–400 nm) of ten species of freshwater and marine phagotrophic protists was assessed in short-term (4 h) laboratory experiments. Changes in the motility and morphology of the cells, as well as direct quantification of DNA damage, were evaluated. The net amount of cyclobutane pyrimidine dimers formed after exposure of the organisms to a weighted dose (Setlow DNA normalized at 300 nm) of 1.7 kJ m⁻² was quantified by an immunoassay using a monoclonal specific antibody directed against thymine dimers (T<>Ts). This is the first application of this method to aquatic protists. The results indicated that marine and freshwater heterotrophic nanoflagellates, representatives from the order Kinetoplastida (Bodo caudatus and Bodo saltans, respectively) accumulate significantly higher DNA damage than protists representatives of the orders Chrysomonadida, Cryptomonadida or Scuticociliatida. The high proportion of A:T bases in the unique kinetoplast DNA, may explain the higher accumulation of T<>Ts found in bodonids. Experiments made with B. saltans to study the dynamics of DNA damage accumulation in the presence of UVR and photorepairing light, indicated that the mechanisms of DNA repair in this species are very inefficient. Furthermore, the dramatic changes observed in the cell morphology of B. saltans probably compromise its recovery. Our results show that sensitivity to UVR among aquatic phagotrophic protists is species-specific and that different cell targets are affected differently among species. While DNA damage in B. saltans was accompanied by motility reduction, altered morphology, and finally mortality, this was not observed in other bodonids as well as in the other species tested.

Tetracapsula bryosalmonae, formerly PKX organism, is a myxozoan parasite that causes proliferative kidney disease in salmonid fish. Its primary hosts, in which it undergoes a sexual phase, are phylactolaemate bryozoans. It develops in the bryozoan coelomic cavity as freely floating sacs which contain two types of cells, stellate cells and sporoplasmogenic cells, which become organised as spores. Eight stellate cells differentiate as four capsulogenic cells and four valve cells which surround a single sporoplasmogenic cell. The sporoplasmogenic cell undergoes meiosis and cytoplasmic fission to produce two sporoplasms with haploid nuclei. Sporoplasms contain secondary cells. The unusual development supports previously obtained data from 18S rDNA sequences, indicating that species of Tetracapsula form a clade. It diverged early in the evolution of the Myxozoa, before the radiation that gave rise to the better known genera belonging to the two orders in the single class Myxosporea. The genus Tetracapsula as seen in bryozoans shares some of the characters unique to the myxosporean phase and others typical of the actinosporean phase of genera belonging to the class Myxosporea. However, it exhibits other features which are not found in either phase. A new class Malacosporea and order Malacovalvulida are proposed to accommodate the family Saccosporidae and genus Tetracapsula. Special features of the new class are the sac-like proliferative body, valve cells not covering the exit point of the polar filament, lack of a stopper-like structure sealing the exit, maintenance of valve cell integrity even at spore maturity, absence of hardened spore walls and unique structure of sporoplasmosomes in the sporoplasms.

In anticipation that improved knowledge of euglenid morphology will provide robust apomorphy-based definitions for clades, transmission and scanning electron microscopy were used to reveal novel morphological patterns associated with the euglenid pellicle. In some taxa, the number of pellicle strips around the cell periphery reduces as discrete whorls at the anterior and posterior ends of the cell. The number of whorls at either end varies between selected euglenid taxa but is invariant within a taxon. The pattern of strip reduction associated with these whorls is shown to have at least three evolutionarily linked states: exponential, pseudoexponential, and linear. Two general equations describe these states near the posterior end of euglenid cells. Exponential patterns of strip reduction near the anterior end are described by a third equation. In addition, several euglenid taxa were found to possess conspicuous pellicle pores. These pores are arranged in discrete rows that follow the articulation zones between adjacent strips. The number of strips between rows of pores varies between taxa and displays a series of consecutive character states that differ by a power of two. The patterns of pores may not only have phylogenetical and taxonomical value but may provide morphological markers for following strip maturation during cytoskeletal reproduction.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by “apofluor”, a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. “Apofluor” includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the “viable” nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by “apofluor”, and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by “apofluor” it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.

Hexamita inflata can derive energy from the degradation of arginine via the arginine dihydrolase pathway. Carbamate kinase catalyses the third enzymatic step of the pathway synthesising ATP from the catabolism of carbamyl phosphate. This study reports the identification and characterisation of a carbamate kinase gene from this free-living diplomonad, together with measurements of carbamate kinase enzyme activity in cell-free extracts and a preliminary analysis of the carbamate kinase mRNA by reverse-transcription polymerase chain reaction. Analysis of the carbamate kinase gene revealed the use of non-canonical codons for glutamine. Phylogenetic studies showed a consistent close relationship between carbamate kinase sequences of H. inflata and Giardia intestinalis.

Preliminary attempts to culture Amoebophrya sp., a parasite of Gymnodinium sanguineum from Chesapeake Bay, indicated that success may be influenced by water quality. To explore that possibility, we determined development time, reproductive output, and infectivity of progeny (i.e. dinospores) for Amoebophrya sp. maintained on G. sanguineum grown in four different culture media. The duration of the parasite's intracellular growth phase showed no significant difference among treatments; however, the time required for completion of multiple parasite generations did, with elapsed time to the middle of the third generation being shorter in nutrient-replete media. Parasites of hosts grown in nutrient-replete medium also produced three to four times more dinospores than those infecting hosts under low-nutrient conditions, with mean values of 380 and 130 dinospores/host, respectively. Dinospore production relative to host biovolume also differed, with peak values of 7.4 per 1,000 μm³ host for nutrient-replete medium and 4.8 per 1,000 μm³ host for nutrient-limited medium. Furthermore, dinospores produced by “high-nutrient” parasites had a higher success rate than those formed by “low-nutrient” parasites. Results suggest that Amoebophrya sp. is well adapted to exploit G. sanguineum populations in nutrient-enriched environments.

An Entamoeba invadens gene encoding a homologue of BiP/GRP78, a 70-kDa heat shock protein or chaperonin was cloned. The predicted E. invadens BiP contained an ATP-binding site, a substrate-recognition domain, and a carboxy-terminal KDEL-peptide. Messenger RNAs of E. invadens for BiP, for a 70-kDa heat shock cognate, for a cyst wall glycoprotein (Jacob), and for chitinase were all induced by heat shock and by encystation medium. The presence of Jacob in heat-shocked amebae was confirmed by confocal microscopy and suggests that heat shock and encystation responses in E. invadens are related.