The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

Triplex real-time PCR for detection of Crithidia mellificae and Lotmaria passim in honey bees.

Currently, light microscopic examination of cell morphology cannot discriminate Crithidia mellificae and Lotmaria passim with 100% certainty. Here, a minor groove-binding (MGB) probe-based multiplex real-time PCR assay was developed for the simultaneous and quantitative detection of C. mellificae and L. passim in honey bees. A conserved Hymenoptera 18S rRNA gene was built in as an internal control that allows accurate detection of PCR inhibition and failure of DNA extraction. The newly developed assay was also applied to field samples. Of 21 honey bee colonies (446 bees) sampled from six counties in both central and eastern Massachusetts, 3 colonies (14.29%) and 8 bees (1.79%) were infected with L. passim, and 1 colony (4.76%) and 1 bee (0.22%) with C. mellificae. Our data showed a low rate of trypanosomatid infection, and L. passim was more prevalent than C. mellificae in honey bee samples in Massachusetts.

The distribution of parasite strains among hosts affects disease spread in a social insect.

Social insects present highly interesting and experimentally amenable systems for the study of disease transmission because they naturally live in dense groups of frequently interacting individuals. Using experimental inoculations of five trypanosomatid strains into groups of its natural host, the bumblebee Bombus terrestris, we investigate the effects of the initial parasite strain distribution across group members on the establishment and transmission success of the different strains to new hosts. For a given number of parasite strains circulating within a host group, transmission to new hosts was increased when the strains were initially inoculated as mixed infections (as opposed to separate single infections), presumably because mixed infections generally favored fast replicating strains. In contrast, separate single infections reduced transmission at least in part through a precedence effect, whereby weak strains appeared to persist by making their host unavailable to superinfection. These results suggest that host groups could benefit from 'compartmentalizing' infections by different parasite strains across different group members, which might be achieved in social insects, for example, by division of labor.

Characterization of Two Species of Trypanosomatidae from the Honey Bee Apis mellifera: Crithidia mellificae Langridge and McGhee, and Lotmaria passim n. gen., n. sp.

Trypanosomatids are increasingly recognized as prevalent in European honey bees (Apis mellifera) and by default are attributed to one recognized species, Crithidia mellificae Langridge and McGhee, 1967. We provide reference genetic and ultrastructural data for type isolates of C. mellificae (ATCC 30254 and 30862) in comparison with two recent isolates from A. mellifera (BRL and SF). Phylogenetics unambiguously identify strains BRL/SF as a novel taxonomic unit distinct from C. mellificae strains 30254/30862 and assign all four strains as lineages of a novel clade within the subfamily Leishmaniinae. In vivo analyses show strains BRL/SF preferably colonize the hindgut, lining the lumen as adherent spheroids in a manner identical to previous descriptions from C. mellificae. Microscopy images show motile forms of C. mellificae are distinct from strains BRL/SF. We propose the binomial Lotmaria passim n. gen., n. sp. for this previously undescribed taxon. Analyses of new and previously accessioned genetic data show C. mellificae is still extant in bee populations, however, L. passim n. gen., n. sp. is currently the predominant trypanosomatid in A. mellifera globally. Our findings require that previous reports of C. mellificae be reconsidered and that subsequent trypanosomatid species designations from Hymenoptera provide genetic support.

Host modulation of parasite competition in multiple infections.

Parasite diversity is a constant challenge to host immune systems and has important clinical implications, but factors underpinning its emergence and maintenance are still poorly understood. Hosts typically harbour multiple parasite genotypes that share both host resources and immune responses. Parasite diversity is thus shaped not only by resource competition between co-infecting parasites but also by host-driven immune-mediated competition. We investigated these effects in an insect-trypanosome system, combining in vivo and in vitro single and double inoculations. In vivo, a non-pathogenic, general immune challenge was used to manipulate host immune condition and resulted in a reduced ability of hosts to defend against a subsequent exposure to the trypanosome parasites, illustrating the costs of immune activation. The associated increase in available host space benefited the weaker parasite strains of each pair as much as the otherwise more competitive strains, resulting in more frequent multiple infections in immune-challenged hosts. In vitro assays showed that in the absence of a host, overall parasite diversity was minimal because the outcome of competition was virtually fixed and resulted in strain extinction. Altogether, this shows that parasite competition is largely host-mediated and suggests a role for host immune condition in the maintenance of parasite diversity.

Molecular divergence defines two distinct lineages of Crithidia bombi (Trypanosomatidae), parasites of bumblebees.

This study provides, for the first time, sequence data for the protozoan flagellates Crithidia bombi and Crithidia mellificae (Kinetoplastea: Trypanosomatidae). We amplified the partial sequences of the small subunit ribosomal RNA (SSU rRNA), glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), cytochrome b (Cyt b), and the complete internal transcribed spacer region 1 (ITS1) of the ribosomal RNA gene region for 66 clones of C. bombi from Switzerland and Alaska. Furthermore, we sequenced the same stretch of SSU rRNA and gGAPDH for one isolate of C. mellificae from Switzerland. All four molecular markers classified the C. bombi samples into two distinct lineages A and B. Both lineages were found in the two sampling locations. Variation within lineages was small or non-existing. Sequence differences between lineages were 1.64% for SSU rRNA, 4.36% for gGAPDH, and 12.02% for Cyt b. The ITS1-sequences of lineages A and B have diverged so much that no alignment was possible. With regard to ITS1, we additionally found fragment length polymorphism (variation in microsatellite repeat numbers) as well as nucleotide diversity within each lineage. Furthermore, the sequences of SSU rRNA and gGAPDH of C. mellificae were different from both lineages of C. bombi. The separation of lineages A and B, based on sequence differences and phylogenetic reconstruction, is so pronounced as to characterize two species of "C. bombi." We propose to retain C. bombi for the more common lineage A and suggest the name Crithidia expoeki n. sp. for lineage B.

Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus crithidia.

Three new species of monoxenous parasites from the Neotropical Heteroptera are described on the basis of the ultrastructure of cells in culture, as well as gene sequences of Spliced Leader (SL) RNA, glyceraldehyde phosphate dehydrogenase (GAPDH) and small subunit (SSU) rRNA. The results have highlighted a striking discrepancy between the morphological (dis)similarities and the phylogenetic affinities among the insect trypanosomatids. Although each of the new species is characterized by a distinct set of morphological characters, based on the predominant promastigotes observed in culture, each of them has been provisionally assigned to the genus Leptomonas pending the future revision of this genus. Yet, instead of the phylogenetic affinity with the other members of this polyphyletic genus, the new species are most closely related to Crithidia species. Thus, the extremely long promastigotes of Leptomonas acus sp. n. and the unique morphological features found in Leptomonas bifurcata sp. n. sharply contrast with their respective relatives C. fasciculata and C. deanei both of which are typical choanomastigotes. The results clearly show that the current classification at the genus level is misleading and needs to be revised. The phylogenetic clades potentially representing the candidate new genera of monoxenous trypanosomatids have started to emerge from the presented analyses.

An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids.

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa.

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.

Differential lectin recognition of glycoproteins in choanomastigote-shaped trypanosomatids: taxonomic implications.

The glycoprotein profiles of seven choanomastigote-shaped trypanosomatids (six Crithidia spp. and one Herpetomonas sp.), which have been suggested to form three distinct taxonomic groups (Crithidia, Angomonas and Strigomonas), were analyzed by Western blotting using the lectins Limax flavus (LFA), Sambucus nigra (SNA) and Maackia amurensis (MAA), which specifically recognize sialic acid residues, and concanavalin A (Con A) that recognizes mannose-like residues in glycoconjugates. All lectins showed a sugar-inhibited recognition with the parasite extracts, with the exception of LFA, which did not show any reactivity with the studied species. The SNA agglutinin presented a characteristic and specific pattern for each taxonomic group. The MAA lectin showed an identical profile for all species analyzed, while Con A grouped the choanomastigote-shaped species in two different patterns, one specific for the Angomonas group, and the other comprehending both Strigomonas and Crithidia groups. These results illustrate the heterogeneity of the genus Crithidia. The possible taxonomic redistribution of the choanomastigote-shaped trypanosomatids is also discussed.

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates.

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.

The heterogeneity of choanomastigote-shaped trypanosomatids as analyzed by their kDNA minicircle size: taxonomic implicaitons.

The kDNA minicircle size was analyzed in 15 species of choanomastigote-shaped trypanosomatids and four main groups of species were identified: (1) "Crithidia" deanei, "C." desouzai and "Herpetomonas" roitmani, which presented the largest molecules (> or = 3,800 bp), (2) "C." oncopelti with minicircles of at least four different sizes within 1,300-2,650 bp, (3) C. fasciculata, C. guilhermei and C. luciliae, having at least two sizes of minicircle (2,650 bp and 3,000 bp) and (4) a heterogeneous group of species presenting minicircles of a single size, including several Crithidia spp. (having 1,600 bp or 1,700 bp minicircles) and two Proteomonas spp. presenting the smallest minicircles (about 1,500 bp). These results were compared with other observations and discussed from a taxonomic point of view.

Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy.

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

Use of glycoconjugates for trypanosomatid taxonomy.

Glycoconjugates from five trypanosomatid genera--Crithidia, Herpetomonas, Endotrypanum, Leishmania, and Trypanosoma--were extracted with Triton X-114 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by periodic acid-Schiff staining. Most of the glycoconjugates were detected in the hydrophobic phase, indicating the presence of anchored glycoconjugates. All the trypanosomatids expressed a glycoconjugate with a low molecular weight (below 20 kDa) in this phase. In each species, however, a characteristic and specific pattern of glycoconjugates was also observed in both phases. In the hydrophobic phase: 14-29 kDa glycoconjugates in C. guilhermei; 24-70 kDa in C. fasciculata, C. luciliae, E. schaudinni, and T. cruzi Y and G strains; 45-66 kDa in C. oncopelti and H. samuelpessoai; above 36 kDa in T. dionisii; 20-24 kDa, 36-45 kDa, and 70 kDa in L. tarentolae and T. mega. In the hydrophilic phase, typical glycoproteins were observed in some trypanosomatids: 60 kDa in T. mega and T. cruzi Y strain; 70 kDa in H. samuelpessoai; 66 kDa in C. oncopelti; 20-70 kDa in C. luciliae. These findings suggest that Triton X-114-extracted glycoconjugates could be useful markers for trypanosomatid taxonomy.

Herpetomonas roitmani (Fiorini et al., 1989) n. comb.: a trypanosomatid with a bacterium-like endosymbiont in the cytoplasm.

The trypanosomatid previously described as Crithidia roitmani is characterized here at the ultrastructural and biochemical levels. The data indicates that the parasite belongs to the Herpetomonas genus, and we therefore suggest the flagellate to be denominated as Herpetomonas roitmani n. comb. Cladistic analysis of isoenzyme data generated by eight different enzymes showed that the parasite presented a distinct banding pattern and could be grouped with some Herpetomonas spp., but not with Crithidia spp., used as reference strains. Accordingly, when the parasites were grown for longer periods in Roitman's defined medium, expontaneous differentiation from promastigotes to opisthomastigotes (typical of the Herpetomonas genus) occurred. Transmission electron microscopy revealed the presence of bacterium-like endosymbionts in the cytoplasm of all evolutive forms of the parasite. All morphological alterations characteristic of endosymbiont-bearing trypanosomatids could be observed.

[Multiple forms of malate dehydrogenase (EC 1.1.1.37) in Crithidia asclepii and C. oncopelti (Mastigophora, Trypanosomatidae)]. / Mnozhestvennye formy malatdegidrogenazy (K.F.1.1.1.37) u Crithidia asclepii i C. oncopelti (Mastigophora, Trypanosomatidae).

Isozyme analysis of mMDH in Crithidia asclepii and C. oncopelti by means of electrophoresis and densimetry method suggests the diploidness and genetic exchange in these organisms. Synthesis of mMDH in C. asclepii is checked by duplicated polymorphic loci MDH-1 and MDH-2, each with two alleles. In C. oncopelti two "daughter" loci are functioning too, polymorphic MDH-1 with 3 alleles and monomorphic MDG-2. Resemblance and differences in the isozyme spectra of mMDH can be used for taxonomic purposes.

Morphologic and biochemical characterization of Crithidia brasiliensis sp. n.

A trypanosomatid with a choanomastigote stage and, therefore, belonging to the genus Crithidia, was isolated in culture from the alimentary tract of the hemipteran genus Zelus. The trypanosomatid was able to grow at 37 C, a characteristic reported to date from only 2 other members of Crithidia, C hutneri and C. luciliae thermophila. Subsequently, the flagellate was cloned for biochemical studies which involved cleaving of kDNA by restriction endonucleases and analyses of the isoenzyme and histone patterns. In all the attributes revealed by the foregoing methods, the organism from Zelus differed from the latter 2 congeneric species. On these and morphologic grounds, this organism appears to belong in a new species for which the name Crithidia brasiliensis sp. n. is proposed.