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Background: Tsetse flies (Diptera: Glossinidae) transmit trypanosomiasis (sleeping sickness in humans and nagana in livestock). Several studies have indicated that age, sex, site of capture, starvation and microbiome symbionts, among others, are important factors that influence trypanosome infection in tsetse flies. However, reasons for a higher infection rate in females than in males still largely remain unknown. Considering that tsetse species and sexes of larger body size are the most mobile and the most available to stationary baits, it was hypothesized in this study that the higher trypanosome prevalence in female than in male tsetse flies was a consequence of females being larger than males., Methods: Black screen fly rounds and Epsilon traps were used to collect tsetse flies in eastern Zambia. Measurement of wing vein length and examination for presence of trypanosomes in the flies were carried out by microscopy. Principal component method was carried out to assess the potential of wing vein length as a predictor variable. The multilevel binary logistic regression method was applied on whole data, one-method data and one-sex data sets to evaluate the hypothesis., Results: Data derived from a total of 2195 Glossina morsitans morsitans were evaluated (1491 males and 704 females). The wing length variable contributed the highest variance percentage (39.2%) to the first principal component. The variable showed significant influence on prevalence of trypanosomes when the analysis was applied on the whole data set, with the log odds for the prevalence of trypanosomes significantly increasing by 0.1 (P  =  0.032), per unit increase in wing length. Females had higher trypanosome prevalence rates than males, though not always significant. Furthermore, moving from females to males, wing length significantly reduced by 0.2 (P  <  0.0001)., Conclusions: We conclude that wing length is an important predictor variable for trypanosome prevalence in Glossina morsitans morsitans and could partially explain the higher prevalence of trypanosomes in females than in males. However, reasonably representative population data are required for analysis-a serious challenge with the current tsetse sampling methods. Thus, analysis combining data from mobile and stationary methods that include both sexes' data could be useful to verify this hypothesis., (© 2021. The Author(s).)

Introduction: A variety of techniques have been used to control tsetse with varying degrees of success. In a study on the population structure of Glossina fuscipes fuscipes that recovered after a previous vector control trial on two Kenyan islands, it was reported that the average fly size on the intervention islands was significantly smaller than on the none intervention islands and also compared to the size before the intervention. The conclusion was that vector control using tiny targets exerted size selection pressure on the population. The study recommended for further studies and suggested that this phenomenon could be among the reasons why targets used as a sole control method have rare reports of successful elimination of tsetse populations. Therefore, in this paper we report on a study of body size of tsetse flies caught in epsilon traps (as a stationary device) and black screen fly rounds (as a mobile trapping device)., Materials and Methods: The study was carried out in eastern Zambia to test the hypothesis that the body size (measured as wing length) of G. m. morsitans males or females, captured by epsilon traps and fly rounds is the same., Results: A total of 1442 (489 females and 953 males) wing length measurements of G. m. morsitans were used in the analysis. It was established that tsetse flies caught by epsilon traps are on average larger than those caught by fly rounds. The likelihood of a large female or male fly being caught by traps, relative to a small one, significantly increased by 5.088 times (95% CI: 3.138-8.429) and by 2.563 times (95% CI: 1.584-4.148), respectively, p < 0.0001, compared with being caught by fly rounds. The hypothesis was rejected., Conclusion: This study showed that epsilon traps capture significantly larger G. m. morsitans than fly rounds do. Therefore, further research is recommended to verify i) whether the predilection of traps to capture larger flies has an effect on the process of tsetse elimination when targets are used e.g. targets may take longer to reach elimination than if the predilection was not there, ii) whether different results can be obtained on ecogeographic distribution of different sizes of the species if fly rounds are used for sampling instead of epsilon traps. The results from such studies could influence the strategies used in future control operations., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effective control of tsetse flies (Diptera; Glossinidae), the biological vectors of trypanosome parasites that cause human African trypanosomosis and African animal trypanosomosis throughout sub-Saharan Africa, is crucial for the development of productive livestock systems. The degree of genetic isolation of the targeted populations, which indicate reinvasion potential from uncontrolled areas, will be critical to establish a control strategy. Molecular and morphometrics markers were used to assess the degree of genetic isolation between seemingly fragmented populations of Glossina brevipalpis Newstead and Glossina austeni Newstead present in South Africa. These populations were also compared with flies from adjacent areas in Mozambique and Eswatini. For the molecular markers, deoxyribonucleic acid was extracted, a r16S2 Polymerase chain reaction (PCR) was performed and the PCR product sequenced. Nine landmarks were used for the morphometrics study as defined by vein intersections in the right wings of female flies. Generalised Procrustes analyses and regression on centroid size were used to determine the Cartesian coordinates for comparison between populations. Both methods indicated an absence of significant barriers to gene flow between the G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Sustainable control can only be achieved if implemented following an area-wide management approach against the entire G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Limited gene flow detected between the G. austeni population from Eswatini and that of South Africa or Mozambique may imply that these two populations are in the proses of becoming isolated.

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

Objective: This study aimed to identify isolated population(s) of Glossina palpalis in Ghana using geometric morphometrics to evaluate variations in wing-shape and size between populations of the fly from three regions., Results: Wing shape of G. palpalis tsetse flies from the Northern, Western and Eastern Regions varied significantly between each other. Populations from the Northern and Western Regions varied the most (Mahalanobis Distance = 54.20). The least variation was noticed between populations from the Western and Eastern Regions (MD = 1.99). On morphospace, the Northern population clearly separated from the Eastern and Western populations both of which overlapped. Wing centroid size also significantly varied among populations. Reclassification scores were satisfactory reaching 100% for the Northern population. The Northern population of G. palpalis is possibly isolated from the Western and Eastern Region populations. Meanwhile, a panmictic relationship could be on-going between the Western and Eastern populations. We speculate that geographical distance and subspecific difference between populations are among factors responsible for observed pattern of wing shape variations among the studied populations. The implications of results regarding choice of control strategy and limitations of the study are discussed.

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.

This is the first study to explore the potential of various geometric morphometrics methods to help the morphological diagnostic of tsetse species, vectors of human and animal trypanosomiases in sub-Saharan Africa. We compared landmarks, semilandmarks and outlines techniques on male and female samples of species, and suggested adapted strategies according to the countries and their own Glossina fauna. We could compare up to 7 taxa belonging to the three main subgenera of the Glossina genus: Nemorhina (5 species), Glossina (1 species) and Austenina (1 species). Our sample included the major vectors of sleeping sickness: G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes and G. f. quanzensis, as well as two important vectors of African animal trypanosomoses: G. tachinoides and Glossina morsitans submorsitans. The average level of correct species recognition by the wing shape was satisfactory, and slightly higher for females than for males. The best scores of correct assignment, in both sexes, were obtained by the contour technique (96% of correct attribution in females, 92% in males), slightly higher than for semilandmarks (95% and 91%) or landmarks (94% and 89%) techniques. We made our images of wings freely available to be used as reference images (http://mome-clic.com), and we describe the conditions and the analytical steps to be followed to identify unknown specimens using external reference images. Under adequate conditions, such use of reference images obtained from a free access server could help species identification of new samples anywhere in Africa., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© 2015 The Royal Entomological Society.)

Neuropeptides and peptide hormones are essential signaling molecules that regulate nearly all physiological processes. The recent release of the tsetse fly genome allowed the construction of a detailed in silico neuropeptide database (International Glossina Genome Consortium, Science 344, 380-386 (2014)), as well as an in-depth mass spectrometric analysis of the most important neuropeptidergic tissues of this medically and economically important insect species. Mass spectrometric confirmation of predicted peptides is a vital step in the functional characterization of neuropeptides, as in vivo peptides can be modified, cleaved, or even mispredicted. Using a nanoscale reversed phase liquid chromatography coupled to a Q Exactive Orbitrap mass spectrometer, we detected 51 putative bioactive neuropeptides encoded by 19 precursors: adipokinetic hormone (AKH) I and II, allatostatin A and B, capability/pyrokinin (capa/PK), corazonin, calcitonin-like diuretic hormone (CT/DH), FMRFamide, hugin, leucokinin, myosuppressin, natalisin, neuropeptide-like precursor (NPLP) 1, orcokinin, pigment dispersing factor (PDF), RYamide, SIFamide, short neuropeptide F (sNPF) and tachykinin. In addition, propeptides, truncated and spacer peptides derived from seven additional precursors were found, and include the precursors of allatostatin C, crustacean cardioactive peptide, corticotropin releasing factor-like diuretic hormone (CRF/DH), ecdysis triggering hormone (ETH), ion transport peptide (ITP), neuropeptide F, and proctolin, respectively. The majority of the identified neuropeptides are present in the central nervous system, with only a limited number of peptides in the corpora cardiaca-corpora allata and midgut. Owing to the large number of identified peptides, this study can be used as a reference for comparative studies in other insects. Graphical Abstract ᅟ.

Background: The biology of adult tsetse (Glossina spp), vectors of trypanosomiasis in Africa, has been extensively studied - but little is known about larviposition in the field., Methodology/principal Findings: In September-November 1998, in the hot-dry season in Zimbabwe's Zambezi Valley, we used artificial warthog burrows to capture adult females as they deposited larvae. Females were subjected to ovarian dissection and were defined as perinatal flies, assumed to have entered burrows to larviposit, if oocyte sizes indicated >95% pregnancy completion. Perinatal flies were defined as full-term pregnant if there was a late third instar larva in utero, or postpartum if the uterus was empty. All other females were defined as pre-full-term pregnant (pre-FT). Of 845 G. m. morsitans captured, 91% (765) were female and 295/724 (41%) of females dissected were perinatal flies. By contrast, of 2805 G. pallidipes captured only 71% (2003) were female and only 33% (596/1825) of females were perinatal. Among all perinatal females 67% (596/891) were G. pallidipes. Conversely, in burrows not fitted with traps - such that flies were free to come and go - 1834 (59%) of pupae deposited were G. m. morsitans and only 1297 (41%) were G. pallidipes. Thus, while more full-term pregnant G. pallidipes enter burrows, greater proportions of G. m. morsitans larviposit in them, reflecting a greater discrimination among G. pallidipes in choosing larviposition sites. Catches of males and pre-FT females increased strongly with temperatures above 32°C, indicating that these flies used burrows as refuges from high ambient temperatures. Conversely, catches of perinatal females changed little with maximum temperature but declined from late September through November: females may anticipate that burrows will be inundated during the forthcoming wet season. Ovarian age distributions of perinatal and pre-FT females were similar, consistent with all ages of females larvipositing in burrows with similar probability., Conclusions/significance: Artificial warthog burrows provide a novel method for collecting tsetse pupae, studying tsetse behaviour at larviposition, assessing the physiological status of female tsetse and their larvae, and of improving understanding of the physiological dynamics of terminal pregnancy, and population dynamics generally, with a view to improving methods of trypanosomiasis control.

Tsetse flies, like many insects, use pheromones for inter- and intra-specific communication. Several of their pheromones are cuticular hydrocarbons (CHCs) that are perceived by contact at close range. We hypothesized that for a successful implementation of the Sterile Insect Technique (SIT), along with proper identification of target area and target species, the target tsetse populations and the sterile flies must chemically communicate with each other. To study the population structuring of Glossina pallidipes in Ethiopia, CHCs were extracted and analyzed from three tsetse belts. As a comparative approach, wing morphometric analysis was performed. The analysis of the relative abundance of CHCs revealed that populations of G. pallidipes from the Rift Valley tsetse belt showed a distinct clustering compared to populations from the other two belts. The spatial pattern of CHC differences was complemented by the wing morphometric analysis. Our data suggest that CHCs of known biological and ecological role, when combined with wing morphometric data, will provide an alternative means for the study of population structuring of Glossina populations. This could aid the planning of area wide control strategies using SIT, which is dependent on sexual competence., (Copyright © 2014 International Atomic Energy Agency 2014. Published by Elsevier B.V. All rights reserved.)

Tsetse flies survive in a variety of environments across tropical Africa, often rising to large numbers, despite their low birth rate of one offspring every seven to nine days. They use olfactory receptors to process chemical signals in their environments to find food, escape from predators, and locate suitable larviposition sites. We discuss the identification of odorant and gustatory receptors in Glossina morsitans morsitans and the role genomics could play in management of nuisance insects., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Sleeping sickness, transmitted by G. p. palpalis, is known to be present in the Ivory Coast. G. p. palpalis has recently been reported to occur in several places within the town of Abidjan, including: (i) the Banco forest, (ii) the Abobo Adjamé University campus and (iii) the zoological park. Could these three places be treated sequentially, as separate tsetse populations, or should they be taken as one area comprising a single, panmictic population?, Methods: The amount of gene flow between these places provides strategic information for vector control. It was estimated by the use of both microsatellite DNA and morphometric markers. The idea was to assess the interest of the faster and much less expensive morphometric approach in providing relevant information about population structure. Thus, to detect possible lack of insect exchange between these neighbouring areas of Abidjan, we used both genetic (microsatellite DNA) and phenetic (geometric morphometrics) markers on the same specimens.Using these same markers, we also compared these samples with specimens from a more distant area of south Ivory Coast, the region of Aniassué (186 km north from Abidjan)., Results: Neither genetic nor phenetic markers detected significant differentiation between the three Abidjan G. p. palpalis samples. Thus, the null hypothesis of a single panmictic population within the city of Abidjan could not be rejected, suggesting the control strategy should not consider them separately. The markers were also in agreement when comparing G. p. palpalis from Abidjan with those of Aniassué, showing significant divergence between the two sites., Conclusions: Both markers suggested that a successful control of tsetse in Abidjan would require the three Abidjan sites to be considered together, either by deploying control measures simultaneously in all three sites, or by a continuous progression of interventions following for instance the "rolling carpet" principle. To compare the geometry of wing venation of tsetse flies is a cheap and fast technique. Agreement with the microsatellite approach highlights its potential for rapid assessment of population structure.

Feeding host is an important factor upon which depend the Glossina infection rate and the proportion of different species of trypanosome. Glossina feed both upon animals and humans. In order to identify species of trypanosomes present in the Komo-Mondah focus and to verify whether there is any relationship between the prevalence of sleeping sickness and the feeding habits of Glossina, we have carried out an entomological survey in this focus of Gabon. Flies were dissected and organs were analysed by PCR, while the origin of blood meals was determined by ELISA. Three species of trypanosomes were found: Trypanosoma congolense "forest type" (14/104; 13.46%), Trypanosoma vivax (11/104; 10.58%) and Trypanosoma brucei s.l. (65/104; 62.5%) with 13.46% (14/104) of mixed infections of T. brucei s.l. and T. congolense Glossina palpalis palpalis was caught in all biotopes investigated (91.85%) and was infected by all these species of trypanosomes. Glossina caliginea was not infected and Glossina fuscipes fuscipes was infected by T. brucei s.l. Tsetse flies feed more on animals than on humans in almost all villages, but there was no significant difference between the number of blood meals taken upon these two groups of vertebrates (Chi 2 = 7.43; p > 0.05). A negative correlation was found between the zoophylic/anthropophylic index and the prevalence of HAT. This result is insufficient to conclude that this index can be used as an indicator of the degree of prevalence of HAT. In contrary, the trypanosomian risk seems to be an appropriate indicator of the prevalence of HAT in an area. The identification of the reservoir hosts in this focus would be useful for a good understanding of the HAT epidemiology.

One of the most sweeping of all patterns in morphological evolution is that animal genitalia tend to diverge more rapidly than do other structures. Abundant indirect evidence supports the cryptic female choice (CFC) explanation of this pattern, which supposes that male genitalia often function to court females during copulation; but direct experimental demonstrations of a stimulatory function have been lacking. In this study, we altered the form of two male genital structures that squeeze the female's abdomen rhythmically in Glossina pallidipes flies. As predicted by theory, this induced CFC against the male: ovulation and sperm storage decreased, while female remating increased. Further experiments showed that these effects were due to changes in tactile stimuli received by the female from the male's altered genitalia, and were not due to other possible changes in the males due to alteration of their genital form. Stimulation from male genital structures also induces females to permit copulation to occur. Together with previous studies of tsetse reproductive physiology, these data constitute the most complete experimental confirmation that sexual selection (probably by CFC) acts on the stimulatory properties of male genitalia.

Tympanal hearing organs are widely used by insects to detect sound pressure. Such ears are relatively uncommon in the order Diptera, having only been reported in two families thus far. This study describes the general anatomical organization and experimentally examines the mechanical resonant properties of an unusual membranous structure situated on the ventral prothorax of the tsetse fly, Glossina morsitans (Diptera: Glossinidae). Anatomically, the prosternal membrane is backed by an air filled chamber and attaches to a pair of sensory chordotonal organs. Mechanically, the membrane shows a broad resonance around 5.3-7.2 kHz. Unlike previously reported dipteran tympana, a directional response to sound was not found in G. morsitans. Collectively, the morphology, the resonant properties and acoustic sensitivity of the tsetse prothorax are consistent with those of the tympanal hearing organs in Ormia sp. and Emblemasoma sp. (Tachinidae and Sarcophagidae). The production of sound by several species of tsetse flies has been repeatedly documented. Yet, clear behavioural evidence for acoustic behaviour is sparse and inconclusive. Together with sound production, the presence of an ear-like structure raises the enticing possibility of auditory communication in tsetse flies and renews interest in the sensory biology of these medically important insects.

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.

A key process in the tsetse reproductive cycle is the transfer of essential nutrients and bacterial symbionts from mother to intrauterine offspring. The tissue mediating this transfer is the milk gland. This work focuses upon the localization and function of two milk proteins (milk gland protein (GmmMGP) and transferrin (GmmTsf)) and the tsetse endosymbionts (Sodalis and Wigglesworthia), in the context of milk gland physiology. Fluorescent in situ hybridization (FISH) and immunohistochemical analysis confirm that the milk gland secretory cells synthesize and secrete milk gland protein and transferrin. Knockdown of gmmmgp by double stranded RNA (dsRNA) mediated RNA interference results in reduction of tsetse fecundity, demonstrating its functional importance in larval nutrition and development. Bacterial species-specific in situ hybridizations of milk gland sections reveal large numbers of Sodalis and Wigglesworthia within the lumen of the milk gland. Sodalis is also localized within the cytoplasm of the secretory cells. Within the lumen, Wigglesworthia localize close to the channels leading to the milk storage reservoir of the milk gland secretory cells. We discuss the significance of the milk gland in larval nutrition and in transmission of symbiotic bacteria to developing offspring.

If species-specific male genitalia are courtship devices under sexual selection by cryptic female choice, then species-specific aspects of the morphology and behaviour of male genitalia should often function to stimulate the female during copulation. The morphology and behaviour of the complex, species-specific male genitalia of the tsetse fly, Glossina pallidipes Austen, were determined from both direct observations and dissections of flash-frozen copulating pairs; we found that some male genitalic traits probably function to stimulate the female, while others function to restrain her. The male clamps the ventral surface of the female's abdomen tightly with his powerful cerci. Clamping does not always result in intromission. Clamping bends the female's body wall and her internal reproductive tract sharply, posteriorly and dorsally, and pinches them tightly. The male performed sustained, complex, stereotyped, rhythmic squeezing movements with his cerci that were not necessary to mechanically restrain the female and appeared instead to have a stimulatory function. Six different groups of modified setae on and near the male's genitalia rub directly against particular sites on the female during squeezing. The designs of these setae correlate with the force with which they press on the female and the probable sensitivity of the female surfaces that they contact. As expected under the hypothesis that these structures are under sexual selection by female choice, several traits suspected to have stimulatory functions have diverged in G. pallidipes and its close relative, G. longipalpis. Additional male non-genitalic behaviour during copulation, redescribed more precisely than in previous publications, is also likely to have a courtship function. The elaborate copulatory courtship behaviour and male genitalia may provide the stimuli that previous studies showed to induce female ovulation and resistance to remating.

The impact of landscape fragmentation due to human and climatic mediated factors on the structure of a population of Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) was investigated in the Mouhoun river basin, Burkina Faso. Allele frequencies at five microsatellite loci, and metric properties based on 11 wing landmarks, were compared between four populations. The populations originated from the Mouhoun river and one of its tributaries. The average distance between samples was 72 km with the two most widely spaced populations being 216 km apart. The sampling points traversed an ecological cline in terms of rainfall and riverine forest ecotype, along a river enlarging from downstream to upstream and oriented south to north. Microsatellite DNA comparison demonstrated structuring between the populations, but not complete isolation, with an overall Fst = 0.012 (P < 0.001). Wing geometry revealed significant centroid size and shape differences between populations, especially between the two most distant populations. There was no significant correlation between gene flow and geographic distance at this scale, but there was a positive correlation in females between metric distances (wing shape differences) and geographic distances that might be attributed to the cline of environmental conditions. The impact of the fragmentation of riparian landscapes on tsetse population structure is discussed in the context of control campaigns currently promoted by Pan African Tsetse and Trypanosomosis Eradication Campaign.

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Allele frequencies at four microsatellite loci, and morphometric features based on 11 wing landmarks, were compared among three populations of Glossina palpalis gambiensis (Diptera: Glossinidae) in Guinea. One population originated from the Loos islands separated from the capital Conakry by 5 km of sea, and the two others originated from the continental mangrove area close to Dubreka, these two groups being separated by approximately 30 km. Microsatellites and wing geometry data both converged to the idea of a separation of the Loos island population from those of the mangrove area. Although occasional contacts cannot be excluded, our results support the hypothesis of the Loos population of tsetse flies being a completely isolated population. This situation will favor a sequenced intervention against human African trypanosomosis and the possibility of an elimination of tsetse from this island.

Tsetse flies transmit the protozoan parasite African trypanosomes, the agents of human sleeping sickness in sub-Saharan Africa. Parasite transmission in the insect is restricted by a natural resistance phenomenon (refractoriness). Understanding the mechanism of parasite resistance is important as strengthening fly's response(s) via transgenic approaches can prevent parasite transmission and lead to the development of novel vector control strategies. Here, we investigated the role of one of the two major pathways regulating innate immunity in invertebrates, the immunodeficiency (Imd) pathway, for Glossina morsitans morsitans's natural defence against Trypanosoma brucei spp. infections. We determined the molecular structure of the Imd pathway transcriptional activator Relish (GmmRel), which shows high amino acid identity and structural similarity to its Drosophila homologue. Through a double-stranded RNA-based interference approach, we showed that the pathogen-induced expression profile of the antimicrobial peptides (AMPs) attacin and cecropin is under the regulation of GmmRel. Unexpectedly, the AMP diptericin appears to be constitutively expressed in tsetse independent of the presence of the Rel factor. Through GmmRel knock-down, we could successfully block the induction of attacin and cecropin expression in the immune responsive tissues fat body and proventriculus (cardia) following microbial challenge. The midgut and salivary gland trypanosome infection prevalence, as well as the intensity of midgut parasite infections were found to be significantly higher in flies when attacin and relish expression were knocked down. Our results provide the first direct evidence for the involvement of antimicrobial peptides in trypanosome transmission in tsetse.

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3' untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised.

Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT > 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.

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.

The size of tsetse flies is often associated with population dynamics and vectorial capacity parameters. Adult fly size is generally estimated from measurements of wing segments. To take measure of the wing, a semi-automatic software was developed by CIRAD-EMVT and IRD. It was used in wild populations of Glossina tachinoides Westwood and G. palpalis gambiensis Vanderplank (Diptera: Glossinidae) trapped near Bobo-Dioulasso, Burkina Faso. From an numeric picture of the wing, the software calculates the length of vein segments, the ratios between these lengths, the surface of the tsetse characteristic "hatchet cell", and the greyness on the wings. The data were interesting at the level of taxonomy. In addition, they help specify physiological characteristics of the studied populations.

Transmission of Trypanosoma brucei by the tsetse fly entails several rounds of differentiation as the parasite migrates through the digestive tract to the salivary glands of its vector. Differentiation of the bloodstream to the procyclic form in the fly midgut is accompanied by the synthesis of a new coat consisting of EP and GPEET procyclins. There are three closely related EP isoforms, two of which (EP1 and EP3) contain N-glycans. To identify the individual EP isoforms that are expressed early during synchronous differentiation in vitro, we exploited the selective extraction of GPI-anchored proteins and mass spectrometry. Unexpectedly, we found that GPEET and all isoforms of EP were coexpressed for a few hours at the onset of differentiation. At this time, the majority of EP1 and EP3 molecules were already glycosylated. Within 24 hours, GPEET became the major surface component, to be replaced in turn by glycosylated forms of EP, principally EP1, at a later phase of development. Transient transfection experiments using reporter genes revealed that each procyclin 3' untranslated region contributes to differential expression as the procyclic form develops. We postulate that programmed expression of other procyclin species will accompany further rounds of differentiation, enabling the parasite to progress through the fly., (Copyright 2001 Academic Press.)

Blood-sucking insects do not all take blood from the host in the same way. Mechanisms are different according to capillary feeding or pool feeding and this explains the differences between clinical reactions. A better knowledge of the insect biology is helpful for an aetiological diagnosis. So, in the case of an allergic reaction, procedures of complementary investigations will be optimised.

Ultrastructural analysis of the corpora cardiaca of the stable fly, Stomoxys calcitrans, and the tsetse fly, Glossina morsitans, revealed the presence of elementary neurosecretory granules (ENG) unique to the intrinsic neurosecretory cells (INC) of these species. In addition to electron-dense spheres, the INC of the corpus species. In addition to electron-dense spheres, the INC of the corpus cardiacum of the stable fly contain electrondense angular granules, either square or rectangular in shape, while the INC of the tsetse fly contain electron-dense spindle-shaped ENG. The distinctive granules of these INC can be traced within nerves to their sites of storage and release, eliminating the need for labeling with artificial probes. Although the INC of the corpus cardiacum of most species have been found to be fuchsinophilic, neither the INC of the stable fly nor the tsetse fly are aldehyde-fuchsinophilic. These peptigenic cells offer neuroendocrinologists a unique opportunity to study the physiology and biochemistry of neurosecretory cells.

The body plan of Drosophila, and presumably that of other insects, develops under the control of anterio-posterior and dorsal ventral axes, but no evidence for a left-right axis has yet been found. We used geometric morphometrics to study the wings in three species of flies: Drosophila melanogaster, Musca domestica and Glossina palpalis gambiensis. In all three species, we found that both size and shape showed subtle, but statistically significant directional asymmetry. For size, these asymmetries were somewhat inconsistent within and between species, but for shape, highly significant directional asymmetry was found in all samples examined. These systematic left-right differences imply the existence of a left-right axis that conveys distinct positional identities to the wing imaginal discs on either body side. Hence, the wing discs of Drosophila may be a new model to study the developmental genetics of left-right asymmetry. The asymmetries of shape were similar among species, suggesting that directional asymmetry has been evolutionarily conserved since the three lineages diverged. We discuss the implications of this evolutionary conservatism in conjunction with results from earlier studies that showed a lack of genetic variation for directional asymmetry in Drosophila.

The effects of maternal age on offspring quality were studied in 1 line of Glossina palpalis palpalis Robineau-Desvoidy, 1 line of G. p. gambiensis Vanderplank, and 3 lines of G. morsitans morsitans Westwood by measuring offspring adult size and the duration of puparial period. G. p. gambiensis males also were examined for effects of maternal age on fluctuating asymmetry of wing veins. The puparial period was shorter in offspring of old females (late offspring) than in offspring of young females (early offspring). The difference was small but was greater for male than for female offspring. Early male offspring were larger than late males. Wing vein fluctuating asymmetry was slightly greater in early than in late offspring in G. p. gambiensis. The differences between early and late offspring were very small, and we conclude that old females produce offspring of marginally lower quality than those produced by young females and that these differences are not biologically significant.

The three-layered peritrophic matrix of Glossina morsitans morsitans is shown, by histochemistry, to be formed of a mixture of glycosaminoglycans, glycoproteins and chitin. In all three layers the glycosaminoglycans contain GlcNAc-hexuronic and Gal-GlcNAc moieties, together with chitin. Glycosaminoglycans in layer 3 are sulphated and sulphated sites have a mean interspace distance of 53 nm - similar to the spacing of fixed charge sites in glomerular basement membrane suggesting a rôle for these sites in the filtration properties of the peritrophic matrix. O-linked oligo- saccharides are present in all three layers. Layer 1 contains the widest variety of glycoprotein oligosaccharide constructs GlcNac and alphalinked GalNAc possibly as GalNAcalpha1,3GalNAc, the latter apparently distal to Galbeta1-4GlcNAc. Lectin binding suggests that layer 2 contains GalNAcalpha1,3Galbeta1,3GlcNAc and that layer 3 contains GalNAc and Galbeta1,4GlcNAc. The evidence for N-linked oligosaccharides is more equivocal. Two dimensional electrophoresis showed that the peritrophic matrix contains a range of proteins, most of which require relatively harsh treatment for their solubilization.

Two methods of salivation of tsetse flies, namely manual salivation and method of Bruce et al. were simultaneously evaluated on 1,702 male uninfected Glossina palpalis palpalis (Zaire), G. palpalis gambiensis (Bobo-Dioulasso), G. p. gambiensis (Maisons-Alfort) et G. morsitans morsitans (Mall) fasted for 23, 48 and 72 hours. The risk of salivation was 0.66 by the manual method and 0.01 by the method of Bruce et al. The manual salivation method was standardised on 79 male G. m. morsitans (Mall) infected with Trypanosoma congolense IL 1180. By this method, 70.88% of flies carrying mature and/or immature infection were identified. A clear difference was observed in the proportion of tsetse flies which salivated after 72 hours and those which salivated after 48 and 24 hours of fasting.

The discovery that tsetse flies are the vectors of African trypanosomosis, causing sleeping sickness in man and nagana in cattle, occurred at the start of a rapidly expanding colonialism in sub-Saharan Africa. Hence, the first research on the fly was largely taxonomic, coupled with a painstaking ecological approach to determine the identities and distribution limits of the different species. This was followed by closer attention to the physiology of the fly, both from the academic standpoint as related to its survival and reproduction in the field, and from the standpoint of its vectorial capacity. There are still conflicting hypotheses concerning the maturation of trypanosomes within the fly. Increasing concern for the environment led to a ban in the developed nations on the use of DDT as an insecticide which had been used successfully for tsetse control in Africa. This was followed by a ban on the use of organochlorine insecticides in general, and no doubt the next restrictions will be on the use of organophosphates and upon synthetic pyrethroids which have already been banned in the UK for the control of houseflies. Fortunately, research on the role of olfactory and visual stimuli of the tsetse, in the location of potential hosts, led to an improvement in methods for monitoring fly populations by means of traps and targets upon which the flies alight. So successful are such devices that, when treated with an insecticide, they can be used to sustain an increase in natural mortality in fly populations to such an extent that these populations decline to manageable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The thoracic eclosion muscles of flies die by cytotoxic attack under neural control. We have investigated the innervation, ultrastructure and immunohistochemistry of the ventral eclosion muscle of Glossina. Two neurons located in the thoracic ganglion innervate this muscle. One of these is immunoreactive for serotonin and does not provide motor innervation. It appears to terminate near the attachment of an immunocyte involved in the dismantling of the muscle. The neuromuscular junction has features that distinguish it from any other chemical junction. A narrow, 3 nm gap separates pre- and post-synaptic membranes and this apparently acts to limit diffusion into and out of the junction. The immunocyte may use neuromuscular innervation as a path-finder to all muscle fibres and may even receive direct input from this source. Neuromuscular transmission is probably chemical as decreasing temperature results in decreasing amplitude of the (graded) muscle potential.

Two species of tsetse flies, Glossina fuscipes fuscipes and G. morsitans submorsitans, are, for the first time, recorded from southwestern Saudi Arabia, near Gizan. This discovery is shortly discussed in relation with the presently known distribution of these species.

The male and female genitalia of Glossina frezili sp. nov., a new tsetse fly occurring in the mangrove forest of the Congo and Gabon are described. G. frezili is related to G. medicorum but clearly distinct from this species by its habitat and the morphology of the genitalia.

The ultrastructural aspects of the nuclear coat formation in midgut epithelial cells of pupae and adult flies of G. morsitans sspp. are described. Out of four different species of Glossina examined, this peculiar structure was only found in G. morsitans sspp. Three different types of viruslike particles were found in midgut epithelial cells. One type which is of the same kind of particle found in the salivary glands, lies inside of cytoplasmic vesicles. Two other types of particles were detected in the nuclei.

A line of Glossina morsitans morsitans Westwood was established in which females have scutellar apical bristles approximately three times as long as normal. In other respects the flies appear normal. The mutant allele, sabr, is recessive to the wild-type allele. The locus for sabr is located in linkage group III, 50 or more map units from the locus for malic dehydrogenase. Scutellar apical bristles in mutant flies are longer in flies emerging from puparia maintained at 30 degrees C than in flies emerging from puparia maintained at 25 degrees C.

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.

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.

The gross morphology of the nervous system supplying the dorsal longitudinal flight muscles of tsetse flies is described. Electron microscopical investigation of the nerves reveals that the dorsal longitudinal muscles are innervated by branches from four main axons. A detailed description of the neuromuscular junctions in the flight muscle is presented.

The study of the mechanism and process of salivation in tsetse flies is of paramount importance towards a fuller understanding of the transmission of trypanosomiasis and nagana diseases. Gordon, Crewe and Willett (1956) made direct observations, through a microscope, of the haustellum of G. morsitans as it penetrated into the ear of an anesthetised mouse and found that probing was accompanied by a copious but intermittent ejection of saliva from the hypopharynx. The outpouring of the saliva commenced during penetration of the stratum corneum and was maintained throughout probing of the tissues. During engorgement, blood is taken in through the labium while saliva was discharged from the hypopharynx at the same time. Besides this work, very little else seems to have been done on the salivary secretion by tsetse flies especially in relation to the hunger cycle of the fly and to the species of tsetse. These aspects were studied on three important species of tsetse and are reported upon in this paper.

The boundary between the distributions of the tsetse sub-species G. palpalis palpalis and G. p. gambiensis lies 60 km north of Bouaké, Ivory Coast. At this place, there is a very narrow hybridization belt. 10 males were caught and compared with hybrids of laboratory origin. The possibility of a postcopulatory sub-specific barrier is discussed.