Signatures of hybridization in Trypanosoma brucei.

Genetic exchange among disease-causing micro-organisms can generate progeny that combine different pathogenic traits. Though sexual reproduction has been described in trypanosomes, its impact on the epidemiology of Human African Trypanosomiasis (HAT) remains controversial. However, human infective and non-human infective strains of Trypanosoma brucei circulate in the same transmission cycles in HAT endemic areas in subsaharan Africa, providing the opportunity for mating during the developmental cycle in the tsetse fly vector. Here we investigated inheritance among progeny from a laboratory cross of T. brucei and then applied these insights to genomic analysis of field-collected isolates to identify signatures of past genetic exchange. Genomes of two parental and four hybrid progeny clones with a range of DNA contents were assembled and analysed by k-mer and single nucleotide polymorphism (SNP) frequencies to determine heterozygosity and chromosomal inheritance. Variant surface glycoprotein (VSG) genes and kinetoplast (mitochondrial) DNA maxi- and minicircles were extracted from each genome to examine how each of these components was inherited in the hybrid progeny. The same bioinformatic approaches were applied to an additional 37 genomes representing the diversity of T. brucei in subsaharan Africa and T. evansi. SNP analysis provided evidence of crossover events affecting all 11 pairs of megabase chromosomes and demonstrated that polyploid hybrids were formed post-meiotically and not by fusion of the parental diploid cells. VSGs and kinetoplast DNA minicircles were inherited biparentally, with approximately equal numbers from each parent, whereas maxicircles were inherited uniparentally. Extrapolation of these findings to field isolates allowed us to distinguish clonal descent from hybridization by comparing maxicircle genotype to VSG and minicircle repertoires. Discordance between maxicircle genotype and VSG and minicircle repertoires indicated inter-lineage hybridization. Significantly, some of the hybridization events we identified involved human infective and non-human infective trypanosomes circulating in the same geographic areas.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly.

Elucidating the mechanism of genetic exchange is fundamental for understanding how genes for such traits as virulence, disease phenotype, and drug resistance are transferred between pathogen strains. Genetic exchange occurs in the parasitic protists Trypanosoma brucei, T. cruzi, and Leishmania major, but the precise cellular mechanisms are unknown, because the process has not been observed directly. Here we exploit the identification of homologs of meiotic genes in the T. brucei genome and demonstrate that three functionally distinct, meiosis-specific proteins are expressed in the nucleus of a single specific cell type, defining a previously undescribed developmental stage occurring within the tsetse fly salivary gland. Expression occurs in clonal and mixed infections, indicating that the meiotic program is an intrinsic but hitherto cryptic part of the developmental cycle of trypanosomes. In experimental crosses, expression of meiosis-specific proteins usually occurred before cell fusion. This is evidence of conventional meiotic division in an excavate protist, and the functional conservation of the meiotic machinery in these divergent organisms underlines the ubiquity and basal evolution of meiosis in eukaryotes.

Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah.

BACKGROUND: In tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS. METHODS: Three fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days. RESULTS: All interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis. CONCLUSIONS: Despite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.

The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement.

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.

Development of the livestock pathogen Trypanosoma (Nannomonas) simiae in the tsetse fly with description of putative sexual stages from the proboscis.

BACKGROUND: Tsetse-transmitted African animal trypanosomiasis is recognised as an important disease of ruminant livestock in sub-Saharan Africa, but also affects domestic pigs, with Trypanosoma simiae notable as a virulent suid pathogen that can rapidly cause death. Trypanosoma simiae is widespread in tsetse-infested regions, but its biology has been little studied compared to T. brucei and T. congolense. METHODS: Trypanosoma simiae procyclics were cultured in vitro and transfected using protocols developed for T. brucei. Genetically modified lines, as well as wild-type trypanosomes, were transmitted through tsetse flies, Glossina pallidipes, to study T. simiae development in the tsetse midgut, proventriculus and proboscis. The development of proventricular trypanosomes was also studied in vitro. Image and mensural data were collected and analysed. RESULTS: A PFR1::YFP line successfully completed development in tsetse, but a YFP::HOP1 line failed to progress beyond midgut infection. Analysis of image and mensural data confirmed that the vector developmental cycles of T. simiae and T. congolense are closely similar, but we also found putative sexual stages in T. simiae, as judged by morphological similarity to these stages in T. brucei. Putative meiotic dividers were abundant among T. simiae trypanosomes in the proboscis, characterised by a large posterior nucleus and two anterior kinetoplasts. Putative gametes and other meiotic intermediates were also identified by characteristic morphology. In vitro development of proventricular forms of T. simiae followed the pattern previously observed for T. congolense: long proventricular trypanosomes rapidly attached to the substrate and shortened markedly before commencing cell division. CONCLUSIONS: To date, T. brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, which occurs in the fly salivary glands. By analogy, sexual stages of T. simiae or T. congolense are predicted to occur in the proboscis, where the corresponding portion of the developmental cycle takes place. While no such stages have been observed in T. congolense, for T. simiae putative sexual stages were abundant in the tsetse proboscis. Although our initial attempt to demonstrate expression of a YFP-tagged, meiosis-specific protein was unsuccessful, the future application of transgenic approaches will facilitate the identification of meiotic stages and hybrids in T. simiae.

Invariant surface glycoprotein 65 of Trypanosoma brucei is a complement C3 receptor.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

Sequential production of gametes during meiosis in trypanosomes.

Meiosis is a core feature of eukaryotes that occurs in all major groups, including the early diverging excavates. In this group, meiosis and production of haploid gametes have been described in the pathogenic protist, Trypanosoma brucei, and mating occurs in the salivary glands of the insect vector, the tsetse fly. Here, we searched for intermediate meiotic stages among trypanosomes from tsetse salivary glands. Many different cell types were recovered, including trypanosomes in Meiosis I and gametes. Significantly, we found trypanosomes containing three nuclei with a 1:2:1 ratio of DNA contents. Some of these cells were undergoing cytokinesis, yielding a mononucleate gamete and a binucleate cell with a nuclear DNA content ratio of 1:2. This cell subsequently produced three more gametes in two further rounds of division. Expression of the cell fusion protein HAP2 (GCS1) was not confined to gametes, but also extended to meiotic intermediates. We propose a model whereby the two nuclei resulting from Meiosis I undergo asynchronous Meiosis II divisions with sequential production of haploid gametes.

Tsetse Fly Transmission Studies of African Trypanosomes.

African trypanosomes are naturally transmitted by bloodsucking tsetse flies in sub-Saharan Africa and these transmission cycles can be reproduced in the laboratory if clean tsetse flies and suitable trypanosomes are available for experiments. Tsetse transmission gives access to more trypanosome developmental stages than are available from in vitro culture, albeit in very small numbers; for example, the sexual stages of Trypanosoma brucei have been isolated from infected tsetse salivary glands, but have not yet been reported from culture. Tsetse transmission also allows for the natural transition between different developmental stages to be studied.Both wild-type and genetically modified trypanosomes have been successfully fly transmitted, and it is possible to manipulate the trypanosome environment inside the fly to some extent, for example, the induction of expression of genes controlled by the Tet repressor by feeding flies with tetracycline.

A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies.

Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.

Fluorescent proteins reveal what trypanosomes get up to inside the tsetse fly.

The discovery and development of fluorescent proteins for the investigation of living cells and whole organisms has been a major advance in biomedical research. This approach was quickly exploited by parasitologists, particularly those studying single-celled protists. Here we describe some of our experiments to illustrate how fluorescent proteins have helped to reveal what trypanosomes get up to inside the tsetse fly. Fluorescent proteins turned the tsetse fly from a "black box" into a bright showcase to track trypanosome migration and development within the insect. Crosses of genetically modified red and green fluorescent trypanosomes produced yellow fluorescent hybrids and established the "when" and "where" of trypanosome sexual reproduction inside the fly. Fluorescent-tagging endogenous proteins enabled us to identify the meiotic division stage and gametes inside the salivary glands of the fly and thus elucidate the mechanism of sexual reproduction in trypanosomes. Without fluorescent proteins we would still be in the "dark ages" of understanding what trypanosomes get up to inside the tsetse fly.

Trypanosoma congolense: In Vitro Culture and Transfection.

Trypanosoma congolense, together with T. vivax and T. brucei, causes African animal trypanosomiasis (AAT), or nagana, a livestock disease carried by bloodsucking tsetse flies in sub-Saharan Africa. These parasitic protists cycle between two hosts: mammal and tsetse fly. The environment offered by each host to the trypanosome is markedly different, and hence the metabolism of stages found in the mammal differs from that of insect stages. For research on new diagnostics and therapeutics, it is appropriate to use the mammalian life cycle stage, bloodstream forms. Insect stages such as procyclics are useful for studying differentiation and also serve as a convenient source of easily cultured, non-infective organisms. Here, we present protocols in current use in our laboratory for the in vitro culture of different life cycle stages of T. congolense-procyclics, epimastigotes, and bloodstream forms-together with methods for transfection enabling the organism to be genetically modified. © 2019 by John Wiley & Sons, Inc.

Fly transmission and mating of Trypanosoma brucei brucei strain 427.

Like yeast, Trypanosoma brucei is a model organism and has a published genome sequence. Although T. b. brucei strain 427 is used for studies of trypanosome molecular biology, particularly antigenic variation, in many labs worldwide, this strain was not selected for the genome sequencing project as it is monomorphic and unable to complete development in the insect vector. Instead, the fly transmissible, mating competent strain TREU 927 was used for the genome project, but is not as easily grown or genetically manipulable as strain 427; furthermore, recent findings have spread concern on the potential human infectivity of TREU 927. Here we show that a 40-year-old cryopreserved line of strain 427, Variant 3, is fly transmissible and also able to undergo genetic exchange with another strain of T. b. brucei. Comparison of Variant 3 with lab isolates of 427 shows that all have variant surface glycoprotein genes 117, 121 and 221, and identical alleles for 3 microsatellite loci. Therefore, despite some differences in molecular karyotype, there is no doubt that Variant 3 is an ancestral line of present day 427 lab isolates. Since Variant 3 grows fast both as bloodstream forms and procyclics and is readily genetically manipulable, it may prove useful where a fly transmissible version of 427 is required.

Asymmetric cell division as a route to reduction in cell length and change in cell morphology in trypanosomes.

African trypanosomes go through at least five developmental stages during their life cycle. The different cellular forms are classified using morphology, including the order of the nucleus, flagellum and kinetoplast along the anterior-posterior axis of the cell, the predominant cell surface molecules and the location within the host. Here, an asymmetrical cell division cycle that is an integral part of the Trypanosoma brucei life cycle has been characterised in further detail through the use of cell cycle stage specific markers. The cell cycle leading to the asymmetric division includes an exquisitely synchronised mitosis and exchange in relative location of organelles along the anterior-posterior axis of the cell. These events are coupled to a change in cell surface architecture. During the asymmetric division, the behaviour of the new flagellum is consistent with a role in determining the location of the plane of cell division, a function previously characterised in procyclic cells. Thus, the asymmetric cell division cycle provides a mechanism for a change in cell morphology and also an explanation for how a reduction in cell length can occur in a cell shaped by a stable microtubule array.

Dynamics of infection and competition between two strains of Trypanosoma brucei brucei in the tsetse fly observed using fluorescent markers.

BACKGROUND: Genetic exchange occurs between Trypanosoma brucei strains during the complex developmental cycle in the tsetse vector, probably within the salivary glands. Successful mating will depend on the dynamics of co-infection with multiple strains, particularly if intraspecific competition occurs. We have previously used T. brucei expressing green fluorescent protein to study parasite development in the vector, enabling even one trypanosome to be visualized. Here we have used two different trypanosome strains transfected with either green or red fluorescent proteins to study the dynamics of co-infection directly in the tsetse fly. RESULTS: The majority of infected flies had both trypanosome strains present in the midgut, but the relative proportion of red and green trypanosome strains varied considerably between flies and between different sections of the midgut in individual flies. Colonization of the paired salivary glands revealed greater variability than for midguts, as each gland could be infected with red and/or green trypanosome strains in variable proportions. Salivary glands with a mixed infection appeared to have a higher density of trypanosomes than glands containing a single strain. Comparison of the numbers of red and green trypanosomes in the proventriculus, salivary exudate and glands from individual flies showed no correlation between the composition of the trypanosome population of the proventriculus and foregut and that of the salivary glands. For each compartment examined (midgut, foregut, salivary glands), there was a significantly higher proportion of mixed infections than expected, assuming the null hypothesis that the development of each trypanosome strain is independent. CONCLUSION: Both the trypanosome strains used were fully capable of infecting tsetse, but the probabilities of infection with each strain were not independent, there being a significantly higher proportion of mixed infections than expected in each of three compartments examined: midgut, proventriculus and salivary glands. Hence there was no evidence of competition between trypanosome strains, but instead co-infection was frequent. Infection rates in co-infected flies were no different to those found routinely in flies infected with a single strain, ruling out the possibility that one strain enhanced infection with the other. We infer that each fly is either permissive or non-permissive of trypanosome infection with at least 3 sequential checkpoints imposed by the midgut, proventriculus and salivary glands. Salivary glands containing both trypanosome strains appeared to contain more trypanosomes than singly-infected glands, suggesting that lack of competition enhances the likelihood of genetic exchange.

Microarchitecture of the tsetse fly proboscis.

BACKGROUND: Tsetse flies (genus Glossina) are large blood-sucking dipteran flies that are important as vectors of human and animal trypanosomiasis in sub-Saharan Africa. Tsetse anatomy has been well described, including detailed accounts of the functional anatomy of the proboscis for piercing host skin and sucking up blood. The proboscis also serves as the developmental site for the infective metacyclic stages of several species of pathogenic livestock trypanosomes that are inoculated into the host with fly saliva. To understand the physical environment in which these trypanosomes develop, we have re-examined the microarchitecture of the tsetse proboscis. RESULTS: We examined proboscises from male and female flies of Glossina pallidipes using light microscopy and scanning electron microscopy (SEM). Each proboscis was removed from the fly head and either examined intact or dissected into the three constituent components: Labrum, labium and hypopharynx. Our light and SEM images reaffirm earlier observations that the tsetse proboscis is a formidably armed weapon, well-adapted for piercing skin, and provide comparative data for G. pallidipes. In addition, the images reveal that the hypopharynx, the narrow tube that delivers saliva to the wound site, ends in a remarkably ornate and complex structure with around ten finger-like projections, each adorned with sucker-like protrusions, contradicting previous descriptions that show a simple, bevelled end like a hypodermic needle. The function of the finger-like projections is speculative; they appear to be flexible and may serve to protect the hypopharynx from influx of blood or microorganisms, or control the flow of saliva. Proboscises were examined after colonisation by Trypanosoma congolense savannah. Consistent with the idea that colonisation commences in the region nearest the foregut, the highest densities of trypanosomes were found in the region of the labrum proximal to the bulb, although high densities were also found in other regions of the labrum. Trypanosomes were visible through the thin wall of the hypopharynx by both light microscopy and SEM. CONCLUSIONS: We highlight the remarkable architecture of the tsetse proboscis, in particular the intricate structure of the distal end of the hypopharynx. Further work is needed to elucidate the function of this intriguing structure.

Trypanosoma congolense: Molecular Toolkit and Resources for Studying a Major Livestock Pathogen and Model Trypanosome.

The African trypanosomiases are diseases of humans and their livestock caused by trypanosomes carried by bloodsucking tsetse flies. Although the human pathogen Trypanosoma brucei is the best known, other trypanosome species are of greater concern for animal health in sub-Saharan Africa. In particular, Trypanosomacongolense is a major cattle pathogen, which is as amenable to laboratory culture as T. brucei, with the advantage that its whole life cycle can be recapitulated in vitro. Thus, besides being worthy of study in its own right, T. congolense could be useful as a model of trypanosome development. Here we review the biology of T. congolense, highlighting significant and intriguing differences from its sister, T. brucei. An up-to-date compilation of methods for cultivating and genetically manipulating T. congolense in the laboratory is provided, based on published work and current development of methods in our lab, as well as a description of available molecular resources.

Dynamics of gamete production and mating in the parasitic protist Trypanosoma brucei.

BACKGROUND: Sexual reproduction in Plasmodium falciparum and Trypanosoma brucei occurs in the insect vector and is important in generating hybrid strains with different combinations of parental characteristics. Production of hybrid parasite genotypes depends on the likelihood of co-infection of the vector with multiple strains. In mosquitoes, existing infection with Plasmodium facilitates the establishment of a second infection, although the asynchronicity of gamete production subsequently prevents mating. In the trypanosome/tsetse system, flies become increasingly refractory to infection as they age, so the likelihood of a fly acquiring a second infection also decreases. This effectively restricts opportunities for trypanosome mating to co-infections picked up by the fly on its first feed, unless an existing infection increases the chance of successful second infection as in the Plasmodium/mosquito system. RESULTS: Using green and red fluorescent trypanosomes, we compared the rates of trypanosome infection and hybrid production in flies co-infected on the first feed, co-infected on a subsequent feed 18 days after emergence, or fed sequentially with each trypanosome clone 18 days apart. Infection rates were highest in the midguts and salivary glands (SG) of flies that received both trypanosome clones in their first feed, and were halved when the infected feed was delayed to day 18. In flies fed the two trypanosome clones sequentially, the second clone often failed to establish a midgut infection and consequently was not present in the SG. Nevertheless, hybrids were recovered from all three groups of infected flies. Meiotic stages and gametes were produced continuously from day 11 to 42 after the infective feed, and in sequentially infected flies, the co-occurrence of gametes led to hybrid formation. CONCLUSIONS: We found that a second trypanosome strain can establish infection in the tsetse SG 18 days after the first infected feed, with co-mingling of gametes and production of trypanosome hybrids. Establishment of the second strain was severely compromised by the strong immune response of the fly to the existing infection. Although sequential infection provides an opportunity for trypanosome mating, the easiest way for a tsetse fly to acquire a mixed infection is by feeding on a co-infected host.

Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor.

The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host.