In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete, Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that:1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13^suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2–3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore “selective digestion” is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.

The merozoite surface protein-1 (MSP-1) is a major vaccine candidate for the asexual blood stage of malaria. We examined both the extent of sequence diversity in block 17, the 3′ end of Msp-1 gene coding for a 19-kDa polypeptide (MSP-1₁₉) putatively involved in red blood cell binding, and the patterns of linkage disequilibrium between polymorphic sites throughout the Msp-1 locus. The parasite population sample consisted of Plasmodium falciparum isolates collected between 1985 and 1998 in Rondônia, an area of hypoendemic malaria transmission in the southwestern Brazilian Amazon. Results were summarized as follows. (1) Seven block-17 sequence variants or haplotypes were found among 130 isolates, including two new haplotypes (novel combinations of previously reported amino acid replacements), here named Brazil-1 (E-TSR-F) and Brazil-2 (Q-TSR-F). (2) As previously shown for other Msp-1 polymorphisms, frequencies of block-17 haplotypes displayed significant temporal variation. (3) Extensive linkage disequilibrium was demonstrated between neighboring dimorphic sites within block 17, as well as between polymorphisms at the 5′ and 3′ ends of Msp-1 (map distance range: 3.83–4.99 kb). (4) The overall patterns of linkage disequilibrium within Msp-1 remained stable over a period of nearly one decade, and examples of possible ‘epidemic’ expansion of parasites carrying particular Msp-1 alleles were found in the 1980s and 1990s. These results are discussed in relation to the population biology of P. falciparum and the development of malaria vaccines based on MSP-1.

Infraciliature and morphogenesis of six rumen ciliates, Ostracodinium mammosum, O. munham, O. dilobum, O. rugoloricatum, O. iwawoi, and O. tiete are described from pyridinated silver carbonate-impregnated specimens. These six Ostracodinium have a similar polybrachykinety arrangement, distinct from that of other ophryoscolecid ciliates and intermediate between those of O. gracile and O. damaliscus. Buccal infraciliature is composed of three polybrachykineties, a kinety loop, and paralabial kineties. Small dorso-adoral polybrachykinety, slender vestibular polybrachykinety, and kinety loop are characteristic. Dorsal infraciliature consists of the dorsal polybrachykinety that extends laterally along the dorsal side of the body. Morphogenesis is different from that of O. gracile, because the right end of ventral primordium extends dorsally to grow into a right primordium without separation.

Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named “O” and “M”, appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the “O” type corresponds to M. refringens and the “M” type to M. maurini.

Concentration and composition of ciliate protozoa in the families Ophryoscolecidae and Isotrichidae were determined in rumen contents of domestic sheep (Ovis ammon aries) from Cyprus. A total of five genera of Ophryoscolecidae were identified, Metadinium, Enoploplastron, Polyplastron, Epidinium, and Ophryoscolex, which included six species: Metadinium affine, Enoploplastron triloricatum, Polyplastron multivesiculatum, Epidinium ecaudatum, Epidinium graini, and Ophryoscolex purkynjei. Eight separate forms of Epidinium were identified (E. ecaudatum f. ecaudatum, E. e. f. caudatum, E. e. f. bicaudatum, E. e. f. tricaudatum, E. e. f. quadricaudatum, E. graini f. graini, E. g. f. caudatricoronatum, and E. g. f. caudaquadricoronatum), along with five forms of Ophryoscolex purkynjei (O. p. f. purkynjei, O. p. f. bifidobicinctus, O. p. f. bifidoquadricinctus, O. p. f. bicoronatus, O. p. f. tricoronatus, and O. p. f. quadricoronatus). Three species of Isotrichidae were observed, Isotricha intestinalis, I. prostoma, and Dasytricha ruminantium. This study reports new host records for three forms of Epidinium graini and Ophryoscolex purkynjei f. bifidobicinctus. The rumen fauna in the family Ophryoscolecidae from Cypriote domestic sheep appear to have limited diversity compared to those from Turkish and Far Eastern (Chinese/ Japanese) sheep, while they are more diverse than those found in Western European (Scottish) and North American (Canadian/Alaskan) sheep.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.

Encephalitozoon hellem is a microsporidian species responsible for opportunistic infections in AIDS patients. Use of a novel chitinase-based method allowed unsheared chromosomal DNA to be recovered from eleven E. hellem isolates derived from three geographic regions. All isolates were typed by 18S rDNA sequencing, which showed that they belonged to internal transcribed spacer type 1. After ethidium bromide staining, pulsed-field gel electrophoresis (PFGE) analysis discriminated two new karyotypes comprising 7 and 8 chromosomal bands respectively, ranging in size from 205- to 272-kb pairs. Genomic size was estimated to be 2.39 Mb. Our data indicate PFGE is useful for typing E. hellem and confirms genetic diversity among E. hellem genotypes.

The abundance and diversity of gymnamoebae in three subsoils varying in compaction and water retention along a 1.2 m transect were documented as the local climatic conditions changed from late summer 1999 through mid-summer 2000. The mean density of gymnamoebae for the loose soil (1,655/g) was greater than either the most compact (1,468/g) or moderately compact soil (851/g). Minimum densities occurred in middle and late summer for all soils while significant (F = 38.803, ≤ 0.0002) density peaks at 3,212/g occurred in early summer in the most compact soil, 2,928/g in the least compact, and 2,209/g in the moderately compact soil. Limax non-eruptive gymnamoebae (mt 2) correlated (r = 0.49, p ≤ 0.016) with moisture while eruptive limax gymnamoebae (mt 3) correlated with temperature (r = 0.07, p ≤ 0.024), moisture (r = 0.58, p ≤ 0.001) and precipitation (r = 0.46, p ≤ 0.029). Flattened or discoid amoebae (mt 4) dominated throughout most of the survey, and the two limax groups showed inverse relationships. Chi-square analyses showed significant differences in the numbers of limax eruptive gymnamoebae compared to all other morphotypes on all but one sampling period.

The cellular organisation of the ‘excavate’ flagellate Jakoba incarcerata Bernard, Simpson and Patterson 2000 is described. Cells have one nucleus and dictyosome. The putative mitochondria lack cristae. Two flagella (anterior and posterior) insert anterior to the feeding groove. The posterior flagellum bears a dorsal vane. An ‘anterior’ microtubular root arises against the anterior basal body. Two main microtubular roots, left and right, and a singlet ‘root’ arise around the posterior basal body and support the groove. Non-microtubular fibres termed ‘A’, ‘B’, ‘I’, and ‘composite’ associate with the right root. A multilaminar ‘C’ fibre associates with the left root. The cytoskeleton of J. incarcerata indicates a common ancestry with other excavate taxa (i.e. diplomonads, retortamonads, heteroloboseids, ‘core jakobids’, Malawimonas, Carpediemonas, and Trimastix). Overall, J. incarcerata is most similar to (other) core jakobids, namely Jakoba libera, Reclinomonas, and Histiona. We regard J. incarcerata as a core jakobid and identify the group by the synapomorphy ‘vanes restricted to dorsal side of the posterior flagellum’. The anterior root and position of the B fibre (and presence of dense inclusions in the cartwheels and a conscpicuous singlet root-associated fibre) in J. incarcerata are novel for core jakobids and argue for close relationships with Trimastix and/or Heterolobosea. The C fibre is similar in substructure to the costal fibre of parabasalids and it is possible that the structures are homologous.

A gene encoding a putative GTP-specific phosphoenolpyruvate carboxykinase has been cloned and sequenced from the type I amitochondriate protist Giardia intestinalis. The deduced amino acid sequence is related most closely to homologs from hyperthermophilic archaebacteria and only more distantly to homologs from Eubacteria and Metazoa. Most enzymes of Giardia core metabolism, however, are related more closely to eubacterial and metazoan homologs. An archaebacterial relationship has been noted previously for the unusual acetyl-CoA synthetase (ADP-forming) of this organism. The results suggest that phosphoenolpyruvate carboxykinase and acetyl-CoA synthetase have been acquired from different sources than most enzymes of Giardia core metabolism.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonas foetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.