Tsetse fly

These are magnified photographs of tsetse flies.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FELLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main chide of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Interactions between trypanosomes and tsetse flies

African trypanosomes are insect-borne parasites that cause sleeping sickness in humans and nagana in domesticated animals. Successful transmission is the outcome of crosstalk between the trypanosome and its insect vector, the tsetse fly. This enables the parasite to undergo successive rounds of differentiation, proliferation and migration, culminating in the infection of a new mammalian host. Several stage- and species-specific parasite surface molecules have been identified and there are new insights into their regulation in the fly. Tsetse flies are often refractory to infection with trypanosomes. While many environmental and physiological factors are known to influence infection, our detailed understanding of tsetse-trypanosome relationships is still in its infancy. Recent studies have identified a number of tsetse genes that show altered expression patterns in response to microbial infections, some of which have also been implicated in modulating trypanosome transmission.

Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

A glycosylation mutant of Trypanosoma brucei links social motility defects in vitro to impaired colonisation of tsetse in vivo

Transmission of African trypanosomes by tsetse flies requires that the parasites migrate out of the midgut lumen and colonise the ectoperitrophic space. Early procyclic culture forms correspond to trypanosomes in the lumen; on agarose plates they exhibit social motility, migrating en masse as radial projections from an inoculation site. We show that an Rft1-/- mutant needs to reach a greater threshold number before migration begins, and that it forms fewer projections than its wild-type parent. The mutant is also up to 4 times less efficient at establishing midgut infections. Ectopic expression of Rft1 rescues social motility defects and restores the ability to colonise the fly. These results are consistent with social motility reflecting movement to the ectoperitrophic space, implicate N-glycans in the signalling cascades for migration in vivo and in vitro, and provide the first evidence that parasite-parasite interactions determine the success of transmission by the insect host.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 107 molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms in vitro. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont, Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related to Escherichia coli, for fertility and possibly nutrition. We show here that the intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for E. coli. We were able to identify 650 orthologous genes in Wigglesworthia corresponding to ≈85% of its genome. The arrays were also applied for expression analysis using Wigglesworthia cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of Wigglesworthia. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that Wigglesworthia may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of E. coli gene arrays to obtain broad genome information for a closely related organism in the absence of complete genome sequence data.

Experimental demonstration of possible cryptic female choice on male tsetse fly genitalia

artículo -- Universidad de Costa Rica. Escuela de Biología, 2009. Este documento es privado debido a restricciones de derechos de autor del publicador.

Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies

Trypanosomiasis has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. Whilst assessments of the biology of trypanosomes, vectors, vertebrate hosts and the environment have provided useful information about life cycles, transmission, and pathogenesis of the parasites that could be used for treatment and control, less information is available about the effects of interactions among multiple intrinsic factors on trypanosome presence in tsetse flies from different sites. It is known that multiple species of tsetse flies can transmit trypanosomes but differences in their vector competence has normally been studied in relation to individual factors in isolation, such as: intrinsic factors of the flies (e.g. age, sex); habitat characteristics; presence of endosymbionts (e.g. Wigglesworthia glossinidia, Sodalis glossinidius); feeding pattern; host communities that the flies feed on; and which species of trypanosomes are transmitted. The purpose of this study was to take a more integrated approach to investigate trypanosome prevalence in tsetse flies. In chapter 2, techniques were optimised for using the Polymerase Chain Reaction (PCR) to identify species of trypanosomes (Trypanosoma vivax, T. congolense, T. brucei, T. simiae, and T. godfreyi) present in four species of tsetse flies (Glossina austeni, G. brevipalpis, G. longipennis and G. pallidipes) from two regions of eastern Kenya (the Shimba Hills and Nguruman). Based on universal primers targeting the internal transcribed spacer 1 region (ITS-1), T. vivax was the predominant pathogenic species detected in flies, both singly and in combination with other species of trypanosomes. Using Generalised Linear Models (GLMs) and likelihood ratio tests to choose the best-fitting models, presence of T. vivax was significantly associated with an interaction between subpopulation (a combination between collection sites and species of Glossina) and sex of the flies (X2 = 7.52, df = 21, P-value = 0.0061); prevalence in females overall was higher than in males but this was not consistent across subpopulations. Similarly, T. congolense was significantly associated only with subpopulation (X2 = 18.77, df = 1, P-value = 0.0046); prevalence was higher overall in the Shimba Hills than in Nguruman but this pattern varied by species of tsetse fly. When associations were analysed in individual species of tsetse flies, there were no consistent associations between trypanosome prevalence and any single factor (site, sex, age) and different combinations of interactions were found to be significant for each. The results thus demonstrated complex interactions between vectors and trypanosome prevalence related to both the distribution and intrinsic factors of tsetse flies. The potential influence of the presence of S. glossinidius on trypanosome presence in tsetse flies was studied in chapter 3. A high number of Sodalis positive flies was found in the Shimba Hills, while there were only two positive flies from Nguruman. Presence or absence of Sodalis was significantly associated with subpopulation while trypanosome presence showed a significant association with age (X2 = 4.65, df = 14, P-value = 0.0310) and an interaction between subpopulation and sex (X2 = 18.94, df = 10, P-value = 0.0043). However, the specific associations that were significant varied across species of trypanosomes, with T. congolense and T. brucei but not T. vivax showing significant interactions involving Sodalis. Although it has previously been concluded that presence of Sodalis increases susceptibility to trypanosomes, the results presented here suggest a more complicated relationship, which may be biased by differences in the distribution and intrinsic factors of tsetse flies, as well as which trypanosome species are considered. In chapter 4 trypanosome status was studied in relation to blood meal sources, feeding status and feeding patterns of G. pallidipes (which was the predominant fly species collected for this study) as determined by sequencing the mitochondrial cytochrome B gene using DNA extracted from abdomen samples. African buffalo and African elephants were the main sources of blood meals but antelopes, warthogs, humans, giraffes and hyenas were also identified. Feeding on multiple hosts was common in flies sampled from the Shimba Hills but most flies from Nguruman had fed on single host species. Based on Multiple Correspondence Analysis (MCA), host-feeding patterns showed a correlation with site of sample collection and Sodalis status, while trypanosome status was correlated with sex and age of the flies, suggesting that recent host-feeding patterns from blood meal analysis cannot predict trypanosome status. In conclusion, the complexity of interactions found suggests that strategies of tsetse fly control should be specific to particular epidemic areas. Future studies should include laboratory experiments that use local colonies of tsetse flies, local strains of trypanosomes and local S. glossinidius under controlled environmental conditions to tease out the factors that affect vector competence and the relative influence of external environmental factors on the dynamics of these interactions.

Phylogenetic analysis of Trypanosoma vivax supports the separation of South American/West African from East African isolates and a new T-vivax-like genotype infecting a nyala antelope from Mozambique

In this study, we addressed the phylogenetic and taxonomic relationships of Trypanosoma vivax and related trypanosomes nested in the subgenus Duttonella through combined morphological and phylogeographical analyses. We previously demonstrated that the clade T. vivax harbours a homogeneous clade comprising West African/South American isolates and the heterogeneous East African isolates. Herein we characterized a trypanosome isolated from a nyala antelope (Tragelaphus angasi) wild-caught in Mozambique (East Africa) and diagnosed as T. vivax-like based on biological, morphological and molecular data. Phylogenetic relationships, phylogeographical patterns and estimates of genetic divergence were based on SSU and ITS rDNA sequences of T. vivax from Brazil and Venezuela (South America), Nigeria (West Africa), and from T. vivax-like trypanosomes from Mozambique, Kenya and Tanzania (East Africa). Despite being well-supported within the T. vivax clade, the nyala trypanosome was highly divergent from all other T. vivax and T. vivax-like trypanosomes, even those from East Africa. Considering its host origin, morphological features, behaviour in experimentally infected goats, phylogenetic placement, and genetic divergence this isolate represents a new genotype of trypanosome closely phylogenetically related to T. vivax. This study corroborated the high complexity and the existence of distinct genotypes yet undescribed within the subgenus Duttonella.

Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports Clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that Circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDII sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.