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Photos of the experimental set-up; GC-MS data of chemical analysis of leaf litter

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly.\ud \ud IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.

While across the animal kingdom offspring are born smaller than their parents, notable exceptions exist. Several dipteran species belonging to the Hippoboscoidea superfamily can produce offspring larger than themselves. In this essay, the blood‐feeding tsetse is focused on. It is suggested that the extreme reproductive strategy of this fly is enabled by feeding solely on highly nutritious blood, and producing larval offspring that are soft and malleable. This immense reproductive expenditure may have evolved to avoid competition with other biting flies. Tsetse also transmit blood‐borne parasites that cause the fatal diseases called African trypanosomiases. It is discussed how tsetse life history and reproductive strategy profoundly influence the type of vector control interventions used to reduce fly populations. In closing, it is argued that the unusual life history of tsetse warrants their preservation in the areas where human and animal health is not threatened.

BACKGROUND\ud Major depressive disorder (MDD) associated with chronic neglected tropical diseases (NTDs) has been identified as a significant and overlooked contributor to overall disease burden. Cutaneous leishmaniasis (CL) is one of the most prevalent and stigmatising NTDs, with an incidence of around 1 million new cases of active CL infection annually. However, the characteristic residual scarring (inactive CL) following almost all cases of active CL has only recently been recognised as part of the CL disease spectrum due to its lasting psychosocial impact.\ud METHODS AND FINDINGS\ud We performed a multi-language systematic review of the psychosocial impact of active and inactive CL. We estimated inactive CL (iCL) prevalence for the first time using reported WHO active CL (aCL) incidence data that were adjusted for life expectancy and underreporting. We then quantified the disability (YLD) burden of co-morbid MDD in CL using MDD disability weights at three severity levels. Overall, we identified 29 studies of CL psychological impact from 5 WHO regions, representing 11 of the 50 highest burden countries for CL. We conservatively calculated the disability burden of co-morbid MDD in CL to be 1.9 million YLDs, which equaled the overall (DALY) disease burden (assuming no excess mortality in depressed CL patients). Thus, upon inclusion of co-morbid MDD alone in both active and inactive CL, the DALY burden was seven times higher than the latest 2016 Global Burden of Disease study estimates, which notably omitted both psychological impact and inactive CL.\ud CONCLUSIONS\ud Failure to include co-morbid MDD and the lasting sequelae of chronic NTDs, as exemplified by CL, leads to large underestimates of overall disease burden.

Background: It has been suggested that attempts to eradicate populations of tsetse (Glossina spp.) using stationary\ud targets might fail because smaller, less mobile individuals are unlikely to be killed by the targets. If true, tsetse\ud caught in stationary traps should be larger than those from mobile baits, which require less mobility on the part\ud of the flies.\ud Results: Sampling tsetse in the Zambezi Valley of Zimbabwe, we found that the number of tsetse caught from\ud stationary traps, as a percent of total numbers from traps plus a mobile vehicle, was ~5% for male G. morsitans\ud morsitans (mean wing length 5.830 mm; 95% CI: 5.800–5.859 mm) and ~10% for females (6.334 mm; 95% CI: 6.329–\ud 6.338 mm); for G. pallidipes the figures were ~50% for males (6.830 mm; 95% CI: 6.821–6.838 mm) and ~75% for\ud females (7.303 mm, 95% CI: 7.302–7.305 mm). As expected, flies of the smaller species (and the smaller sex) were\ud less likely to be captured using stationary, rather than mobile sampling devices. For flies of a given sex and species\ud the situation was more complex. Multivariable analysis showed that, for females of both species, wing lengths\ud changed with ovarian age and the month, year and method of capture. For G. pallidipes, there were statistically\ud significant interactions between ovarian age and capture month, year and method. For G. m. morsitans, there was\ud only a significant interaction between ovarian age and capture month. The effect of capture method was, however,\ud small in absolute terms: for G. pallidipes and G. m. morsitans flies caught on the mobile vehicle had wings only 0.24\ud and 0.48% shorter, respectively, than flies caught in stationary traps. In summary, wing length in field samples of\ud tsetse varies with ovarian age, capture month and year and, weakly, with capture method. Suggestions that a\ud target-based operation against G. f. fuscipes in Kenya caused a shift towards a smaller, less mobile population of\ud tsetse, unavailable to the targets, failed to account for factors other than capture method.\ud Conclusions: The results are consistent with the successful use of targets to eradicate populations of tsetse in\ud Zimbabwe. Until further, more nuanced, studies are conducted, it is premature to conclude that targets alone could\ud not, similarly, be used to eradicate G. f. fuscipes populations in Kenya.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the Variant Surface Glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number and chromosomal position. Thus, analysing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analysing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSG segregate into defined 'phylotypes' that do not recombine. In our dataset comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the 'Variant Antigen Profile'. We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales. [Abstract copyright: Published by Cold Spring Harbor Laboratory Press.]

The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission., Author Summary Blood feeding arthropods are exploited by blood borne parasites as vectors of transmission. Trypanosoma brucei, a salivarian trypanosome species, must survive, migrate and differentiate in the tsetse until they become mature, mammalian-infective forms within the fly salivary glands. This constitutes a significant challenge to trypanosomes as the major parasite form colonising the tsetse midgut is sensitive to lysis by blood complement, which is introduced into the tsetse gut whenever the fly feeds. In this study, we show that T. brucei may avoid being eliminated by bloodmeal complement by benefitting from a complement-inhibiting enzyme secreted by the fly itself. We showed that this serine protease inhibitor (serpin) enzyme, Serpin10, can inactivate triggers of the complement cascade, protect tsetse-infective trypanosomes from complement lysis, and is important for trypanosome establishment in the tsetse midgut. Taken together, we propose that GmmSRPN10 may be part of a repertoire of complement-inhibiting proteins secreted by tsetse that are utilized by T. brucei to evade complement lysis in the tsetse midgut.

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology. ispartof: Science vol:344 issue:6182 pages:380-386 ispartof: location:United States status: published

Tsetse flies are the most important vectors of African trypanosomiasis but, surprisingly, are highly refractory to trypanosome parasite infection. In populations of wild caught flies, it is rare to find mature salivarian and mouthpart parasite infection rates exceeding 1 and 15%, respectively. This inherent refractoriness persists throughout the lifespan of the fly, although extreme starvation and suboptimal environmental conditions can cause a reversion to the susceptible phenotype. The teneral phenomenon is a phenotype unique to newly emerged, previously unfed tsetse, and is evidenced by a profound susceptibility to trypanosome infection. This susceptibility persists for only a few days post-emergence and decreases with fly age and bloodmeal acquisition. Researchers investigating trypanosome-tsetse interactions routinely exploit this phenomenon by using young, unfed (teneral) flies to naturally boost trypanosome establishment and maturation rates. A suite of factors may contribute, at least in part, to this unusual parasite permissive phenotype. These include the physical maturity of midgut barriers, the activation of immunoresponsive tissues and their effector molecules, and the role of the microflora within the midgut of the newly emerged fly. However, at present, the molecular mechanisms that underpin the teneral phenomenon still remain unknown. This review will provide a historical overview of the teneral phenomenon and will examine immune-related factors that influence, and may help us better understand, this unusual phenotype

The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome\ud infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly\ud but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian\ud trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher’s exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral\ud refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed.