Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

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.

Serotonergic Innervation of the Salivary Glands and Central Nervous System of Adult Glossina pallidipes Austen (Diptera: Glossinidae), and the Impact of the Salivary Gland Hypertrophy Virus (GpSGHV) on the Host.

Using a serotonin antibody and confocal microscopy, this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly, Glossina pallidipes Austen. Reports to date, however, note that up until this finding, dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands. Direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host, thus delaying a host response. Our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is due to a compensatory increased branching of the neurons of the salivary glands, which is associated with the increased size of the salivary glands in viral infected flies. This study shows for the first time serotonin in the cell bodies of the brain and thoracico-abdominal ganglion in adult tsetse, G. pallidipes Austen (Diptera: Glossinidae). A hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans; and, a plea is made that more research is needed to develop a blood feeding model, similar to that in the blow flies, for elucidating the various mechanisms involved in production and deployment of saliva.

Disruption of the salivary gland muscle in tsetse, Glossina pallidipes Austen, as a result of salivary gland hypertrophy virus infection.

The secretory region of the salivary glands in Glossina pallidipes Austen (Diptera: Glossinidae) is characterized by an external muscle layer. Scanning electron microscopy and transmission electron microscopy investigations provide a detailed description of the longitudinal muscle fibres and a comparison of their structure when affected by salivary gland hypertrophy virus. The virus is responsible for hypertrophy of the salivary glands in symptomatic flies, specifically of the muscle fibres, the cytoarchitecture of which is completely altered. Although observations did not reveal viral particles in the muscle cells of either asymptomatic or symptomatic flies, muscle fibres were enlarged and detached from one another and their associated basement membrane only in symptomatic flies. A decrease in type IV collagen labelling in the basement membrane of the muscles in symptomatic flies is reported and is considered a potential cause of the salivary gland muscle alteration and, possibly, myopathy. The maintenance of an organized muscular layer is essential for the normal secretion of saliva and hence its pathology in symptomatic tsetse flies could affect the normal transmission of the trypanosome that develops inside the salivary gland epithelium. Therefore, a better understanding of the possible role of the virus is essential in order to elucidate its impact on salivary deployment in symptomatic flies.

Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus.

Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite.

Two hytrosaviruses, MdSGHV and GpSGHV, induce distinct cytopathologies in their respective host insects.

Recently, a new virus family (Hytrosaviridae) was proposed for double-stranded DNA viruses that cause salivary gland hypertrophy in their dipteran hosts. The two type species, MdSGHV and GpSGHV, induce similar gross pathology and share several morphological, biological, and molecular characteristics. This histological study revealed profound differences in the cytopathology of these viruses supporting their previously proposed placement in different genera.

Fine structure of the female reproductive system in a viviparous insect, Glossina morsitans morsitans (Diptera, Glossinidae).

The female reproductive system of the tsetse fly Glossina morsitans morsitans is analysed by scanning electron microscopy (SEM). The study focuses in particular on the choriothete, a peculiar uterine structure involved in the viviparous mode of reproduction of Glossina morsitans morsitans. Under light microscopy, the choriothete appears formed by numerous tongue-like folds projecting towards the uterine lumen and lined by a thin cuticle. SEM analysis highlights for the first time a distinctive new feature that is not visible by traditional histological methods. That is a cuticular covering of the choriothete, which shows numerous thorns in the form of crest-like structures arranged in nearly parallel lines. The role of the choriothete in pregnancy and in larval nourishment is discussed.

The heart of darkness: growth and form of Trypanosoma brucei in the tsetse fly.

The first description of African trypanosomes was made over a century ago. The importance of the tsetse in transmission and cyclic development of trypanosomes was discovered soon afterwards, and has been the focus of numerous studies since. However, investigation of trypanosomes in tsetse flies requires high resource investment and unusual patience; hence, many facets of trypanosome biology in the tsetse remain to be characterised despite the long history of research. Here, current knowledge and questions about some of the developmental changes in trypanosomes that occur in tsetse flies are summarised, along with recent technical advances that can now be used to provide some answers.

TsetseEP, a gut protein from the tsetse Glossina morsitans, is related to a major surface glycoprotein of trypanosomes transmitted by the fly and to the products of a Drosophila gene family.

African trypanosomes live in the lumen of the gut of tsetse (Glossina) and may have to face an immune response. As yet, it is unclear whether they are sensitive to antimicrobial peptides in vivo, but for some years there has been indirect evidence that one or more lectins can influence the infection. We have purified a protein complex from midgut extracts that, by SDS-PAGE, is a doublet of 37 and 38 kDa in a ratio of 3:1. Through prediction from corresponding cDNA clones, the full-length protein (tsetseEP) contains 320 amino acids, including a signal peptide. There is apparently only one gene encoding this protein. Towards the C terminus, the protein contains a run of 59 (EP) repeats, which surprisingly is what comprises almost the entire mature EP procyclin molecule present on the surface of trypanosomes in the tsetse gut. Drosophila contains a number of genes encoding proteins, of unknown function, with the same cysteine pattern as tsetseEP; this pattern is not reported for any other protein. Immunoblotting with a monoclonal antibody against (EP) repeats reveals expression in the gut, but not salivary glands, of female and male flies, whether or not fed. Immunoelectron microscopy shows the presence in vesicles in midgut cells and in the lumen of the gut. Attempts to demonstrate lectin activity were thwarted by limited availability of the protein complex.

Abdominal pericardial sinus: a neurohemal site in the tsetse and other cyclorraphan flies.

An ultrastructural study of the heart of the tsetse fly, Glossina morsitans, and of several other species of cyclorraphan flies revealed that the ventral region of the heart of adult flies is supported by a muscular septum not present in the larval stage. The pericardial septum of the adult heart is composed laterally of alary muscles and a central longitudinal muscle that extends the length of the abdominal aorta, whereas the larval heart is supported ventrally only by alary muscles and strands of connective tissue. Thus, unlike the larval stage, and the heart of other insects, the pericardial septum of adult cyclorraphan flies contains a central band of longitudinal muscle, that along with the alary muscle, forms a large pericardial sinus lying between the septum and the heart. Neurosecretory nerves arising from the lateral nerves of the thoracicoabdominal ganglion extend dorsad to the pericardial septum, where they form neuromuscular junctions on the muscle fibers of the pericardial septum or traverse the septum terminating in the pericardial sinus, thereby creating one of the largest neurohemal organs in these flies. In the tsetse fly, some of the neurosecretory fibers also extend between the muscle fibers of the myocardium, and release their material into the lumen of the heart.

Photographic polytene chromosome maps for Glossina morsitans submorsitans (Diptera: Glossinidae): cytogenetic analysis of a colony with sex-ratio distortion.

Photographic polytene chromosome maps from trichogen cells of pharate adult Glossina morsitans submorsitans were constructed. Using the standard system employed to map polytene chromosomes of Drosophila, the characteristic landmarks were described for the X chromosome and the two autosomes (L1 and L2). Sex-ratio distortion, which is expressed in male G. m. submorsitans, was found to be associated with an X chromosome (X8) that contains three inversions in each arm. Preliminary data indicate no differences in the fecundity of X(A)X(A) and X(A)X(B) females, but there are indications that G. m. submorsitans in colonies originating from Burkina Faso and Nigeria have genes on the autosomes and (or) the Y chromosome that suppress expression of sex-ratio distortion.

Genetic transformation and phylogeny of bacterial symbionts from tsetse.

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia-like organisms reported previously from various tsetse species. The 16S rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti-parasitic genes within tsetse populations.

Ultrastructural changes in salivary glands of tsetse, Glossina morsitans morsitans, infected with virus and rickettsia-like organisms.

Electron microscope observations on enlarged hypertrophied salivary glands dissected from adult laboratory-reared male Glossina morsitans morsitans show a concurrent infection of the salivary gland tissue with rod-shaped virus particles and intracellular rickettsia-like organisms. The latter are found intracellular in the epithelium and in the gland lumen enclosed within lytic zones. The virus particles are found within the degenerating cytoplasm, nuclei, and lumen of the cell where they are especially numerous. Stratified epithelium and gland enlargement are a prominent feature of the infection. These observations suggest that biological associations between salivary gland tissue and diverse microbes may be more common than formerly recognized. The microbes appear to cause damage to salivary gland cells, causing hyperplasia which assumes pathologic proportions.

Some concepts of the interaction of Trypanosoma (Nannomonas) congolense and Glossina pallidipes.

Light and electron microscope investigations were carried out on the infection with Trypanosoma (Nannomonas) congolense of laboratory-reared tsetse flies Glossina pallidipes. Trypanosomes became entombed in the peritrophic membrane (PM) to form intraperitrophic cavities which were more electron-translucent than the amorphous layer of the PM. A hypothesis is suggested that after migration anteriorly in the ectoperitrophic space, the trypanosomes become enmeshed in the PM during its formation in the proventriculus, and that the trypanosomes are extricated in the midgut as the PM advances towards the posterior end of the gut.

In vitro cultivation of rickettsia-like-organisms from Glossina spp.

A method is described for the in vitro cultivation of the rickettsia-like-organisms (RLO) from Glossina spp. which are believed to be associated with susceptibility to trypanosome infection. Cultures of RLO were established by infecting a mosquito cell line (Aedes albopictus) with haemolymph taken from teneral flies. RLO from nine species of Glossina have been isolated and maintained in continuous culture using this technique.

Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat.

Acquisition of the variable antigen-containing surface coat of Trypanosoma brucei occurs at the metacyclic stage in the salivary glands of the tsetse fly vector. The differentiation of the metacyclic trypanosome in the gland has been studied by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze-fracture replicas. The uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) divide while attached to the salivary gland epithelium brush border by elaborate branched flagellar outgrowths, which ramify between the host cell microvilli and form punctate hemidesmosome-like attachment plaques where they are indented by the microvilli. These outgrowths become reduced as the epimastigotes transform to uncoated trypomastigotes (with postnuclear kinetoplast), which remain attached and capable of binary fission. The flagellar outgrowths disappear but the attachment plaques persist as the uncoated trypomastigotes (premetacyclics) stop dividing and acquire the surface coat to become 'nascent metacyclics'. Coat acquisition therefore occurs in the attached trypanosome and not, as previously believed, after detachment. Coating is accompanied by morphological changes in the glycosomes and mitochondrion of the parasite. Freeze-fracture replicas of the host-parasite junctional complexes show membrane particle aggregates on the host membrane but not on the parasite membrane. It is suggested that disruption of the complex occurs when maximum packing of the glycoprotein molecules has been achieved in the trypanosome surface coat, releasing the metacyclic trypanosome into the lumen of the gland.

Notes on midgut cell nuclear coats in various tsetse species.

Coats were found on the midgut cell nuclei of G.m. morsitans, G. austeni, G. tachinoides, G. f. fuscipes and G. p. palpalis. No coat was found in G. p. gambiensis. The coats were of differing ultrastructural design and of different dimensions in each species. The appearance of the coat seems to be linked to the physiological train of events following the bloodmeal rather than to novel events such as viral or protozoal infection. The timing of its appearance varied among the different species examined.