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.

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.

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.

Virus-like rods associated with salivary gland hyperplasia in tsetse, Glossina pallidipes.

Long, virus-like rods were discovered in hyperplastic salivary glands of Glossina pallidipes Austen from Kibwezi Forest (2 degrees 27' S, 37 degrees 55' E), Kenya. The glands were enlarged up to four times the normal diameter. This increase in size was due to a cellular proliferation of the glandular epithelial cells and hypertrophy of their nuclei and cytoplasm. Nuclear and cytoplasmic inclusions were present in the enlarged cells but were not found in cells of normal-sized glands. Electron microscopy revealed many virus-liked rods in the abnormal glands. Males with such glands were often completely sterile. Abnormal growth of the ovarioles was a significant feature of young females with hyperplastic glands. Both sexes of wild and laboratory-bred flies were found with enlarged glands. One way of transmission of the trait seems to be from mother to progeny.

The micro-organisms of tsetse flies.

Micro-organisms from tsetse fly mycetomes were maintained in culture, where they were more pleomorphic than in the mycetomes, but were in some cases very similar to those observed in ovaries by other authors. Agglutination tests on the cultured forms indicated in affinity to Rickettsia. They were sensitive to antibiotics introduced by feeding flies on hosts treated with Ampicillin; this reduced the longevity and fecundity of the tsetse flies and appeared to disturb normal digestion of bloodmeals.

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.

Structural and functional dynamics of oogenesis in Glossina austeni: vitellogenesis with special reference to the follicular epithelium.

Morphological changes of the oocyte, follicle cells and nurse cells of the ovaries of the viviparous fly Glossina austeni during vitellogenesis and postvitellogenesis are outlined. During vitellogenesis, material is pinocytosed and incorporated into yolk spheres by subsequent fusions. Various lines of evidence are presented that indicate much of this material is derived from the follicular epithelium. The ultrastructure of the follicular cells throughout the 9 day cycle and their role in protein synthesis is presented. Subsequent to vitellogenesis, the follicle cells synthesize the secondary envelopes.

Structural and functional dynamics of oogenesis in Glossina austeni: general features, previtellogenesis and nurse cells.

Glossina austeni oogenesis throughout its nine-day pregnancy cycle is described with the focus on previtellogenic stages. The ultrastructural details of the oocyte-nurse cell relationship and cyst formation is presented. The oocyte develops in a syncytial association with 15 nurse cells with the entire unit surrounded by a follicular epithelium. The nurse cells have large elaborate nucleoli. Evidence of nuclear emissions and the presence of an unusual cytoplasmic membrane association were found. A variety of nuclear inclusions are seen in the oocyte. Glycogen, lipid, ribosomes and membrane organelles accumulate in the oocyte during previtellogenesis.

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.

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.

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.