Molecular phylogenetics of tsetse flies (Diptera: Glossinidae) based on mitochondrial (COI, 16S, ND2) and nuclear ribosomal DNA sequences, with an emphasis on the palpalis group

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.     

Host preferences of tsetse (Diptera: Glossinidae) based on bloodmeal identifications

An enzyme-linked immunosorbent assay (ELISA) was developed to identify the origin of vertebrate blood in the guts of 29 245 wild-caught flies of eleven Glossina species from various ecological zones of Africa. Depending on the quality of the bloodmeal samples, 62.8% of the samples were identified and could be assigned to a host-group (e.g. ruminant), family (e.g. Bovidae) or species (e.g. Bos spp.). A total of 13 145 samples (44.9%) was identifiable up to the species level. With a few exceptions, the present results are in agreement with earlier published reports. Glossina austeni and G. fuscipleuris seemed to have a distinct feeding preference for Suidae (mainly bushpig). Glossina morsitans ssp. fed mainly on Suidae (mainly warthog), although local variations were observed and in some areas hippopotamus or ruminants replaced the warthog as the main host. Bushbuck seemed to be the principal food source for G. longipalpis and G. fusca . Glossina pallidipes fed mainly on ruminants (buffalo, bushbuck and cattle) but, depending on host availability and location, Suidae were also important hosts. Hippopotamus was identified as the main source of blood-meals for G. brevipalpis . The main hosts for G. longipennis were Suidae (mainly bushpig) and not rhinoceros as had been reported 40 years earlier. The opportunistic feeding behaviour of the palpalis tsetse group was confirmed. The results showed that changes in environment, fauna and host availability may result in modification of tsetse feeding patterns.     

Phylogeny of genus Glossina (Diptera: Glossinidae) according to ITS2 sequences

The flies of genus Glossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from different Glossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genus Glossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genus Glossina where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest that Glossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with the morsitans group species.     

Comparative study on the infection rates of different laboratory strains of Glossina species by Trypanosoma congolense

Teneral Glossina morsitans centralis Machado, G.austeni Newstead, G.palpalis palpalis Robineau-Desvoidy, G.p.gambiensis Vanderplank, G.fuscipes fuscipes Newstead, G.tachinoides Westwood and G.brevipalpis Newstead, from laboratory-bred colonies, were fed at the same time on the flanks of ten goats infected with Trypanosoma congolense Broden isolated in Tanzania or in Nigeria. The seven tsetse species were infected over the range 0.3-49.2%. Survival of both T.congolense isolates was best in G.m.centralis, poorest in G.austeni and the four palpalis group tsetse, with G.brevipalpis intermediate. It is suggested that there are differences in the gut of different laboratory-bred cultures of Glossina Westwood species and subspecies such that T.congolense parasites can survive better in the gut of some than in others and undergo cyclical development to metacyclics in the hypopharynx.     

Midgut lectin activity and sugar specificity in teneral and fed tsetse

Abstract. . Midgut infection rates of Trypanosoma congolense in Glossina palpalis palpalis and of Trypanosoma brucei rhodesiense in Glossina pallidipes are potentiated by the addition of D+ glucosamine to the infective feed, but not to the levels of super-infection reported for G. m. morsitans. G. p. palpalis and G.pallidipes are shown to possess two trypanocidal molecules: a glucosyl lectin which can be inhibited by D+ glucosamine and a galactosyl molecule inhibited by D+ galactose. Addition of both D+ glucosamine and D+ galactose to the teneral infective feed promotes super-infection of the midguts of G.p.palpalis. The glucosyl lectin is specific for rabbit erythrocytes and is present in guts of fed G.m.morsitans and G.p.palpalis , titres of lectin activity do not increase substantially after the second bloodmeal. The galactosyl specific molecule does not show any erythrocyte specificity, although haemolytic activity is observed only in G.p.palpalis and not in G.m.morsitans. The presence of two trypanocidal molecules in some species of tsetse may account for the innate refractoriness of these flies to trypanosome infection.
As D+ glucosamine also inhibits the killing of procyclic trypanosomes taken as an infective feed, it is suggested that the midgut lectin is normally responsible for the agglutination of trypanosomes in the fly midgut by binding to the pro-cyclic surface coat, prior to establishment in the ecto-peritrophic space.     

Detection and characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in laboratory and natural populations of different species of tsetse flies (genus <Emphasis Type="Italic">Glossina</Emphasis>)

BACKGROUND: Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. RESULTS: In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome. CONCLUSIONS: Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.     

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.     

Functional Morphology of the Phallosome in Glossina (Diptera, Glossinidae) and Its Evolutionary Implications

Current views on the phylo-genetjc relations of the three species groups within the genus Glossina are critically reviewed. In respect of the male genitalia, the fusca group is here regarded as more specialized than the morsitans group. The detailed phallosome morphology of G. austeni is compared with that of G. fuscipes , on the basis of their mode of action in living flies. The comparison is extended to fusca group species, using dead material only. The presumed phylogeny of Glossina is presented in outline; the evolution of the genus has involved a progressive invasion of riverine and later of forest habitats, from an original semi-arid grassy woodland habitat.     

Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types

Abstract Teneral Glossina morsitans mositans, G.m.submorsitans, G.palpalis gambiensis and G.tachinoides were allowed to feed on rabbits infected with Trypanosoma congolense savannah type or on mice infected with T.congolense riverine-forest type. The four tsetse species and subspecies were also infected simultaneously in vitro on the blood of mice infected with the two clones of T.congolense via a silicone membrane. The infected tsetse were maintained on rabbits and from the day 25 after the infective feed, the surviving tsetse were dissected in order to determine the infection rates.
Results showed higher mature infection rates in morsitans-gwup tsetse flies than in palpalis-group tsetse flies when infected with the savannah type of T.congolense. In contrast, infection rates with the riverine-forest type of T.congolense were lower, and fewer flies showed full development cycle. The intrinsec vectorial capacity of G.m.submorsitans for the two T.congolense types was the highest, whereas the intrinsic vectorial capacity of G.p.gambiensis for the Savannah type and G.m.morsitans for the riverine-forest type were the lowest. Among all tsetse which were infected simultaneously with the two types of T.congolense , the polymerase chain reaction detected only five flies which had both trypanosome taxa in the midgut and the proboscis. All the other infections were attributable to the savannah type.
The differences in the gut of different Glossina species and subspecies allowing these two sub-groups of T.congolense to survive better and undergo the complete developmental cycle more readily in some species than other are discussed.     

Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense Savannah Type

Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossina morsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont was investigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts of both fly species, and into the probosces of flies displaying either mature or immature infection, to investigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in probosces from Glossina palpalis gambiensis flies displaying mature or immature infection, but never in probosces from Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlation between the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) the symbiont is probably not involved in Trypanosoma congolense savannah type maturation. It could however participate in the establishment process of the parasite.     

Responses of tsetse flies (Glossina spp.) to compounds on the skin surface of an ox: a laboratory study

The behaviour of male Glossina morsitans morsitans Westwood and Glossina pallidipes Austen (Diptera: Glossinidae) alighting on targets with or without ox sebum was compared. The presence of ox sebum did not increase significantly the number of flies alighting on the target in either species. However, after contact with the sebum coated target, both species showed an increase in flight activity, and G. m.morsitans showed a greater tendency to return to the target. This behaviour resulted in a number of short flights which may reflect the search for a feeding site on a host. The duration of each visit to the target was significantly reduced when sebum was present for G. m. morsitans but not for G. pallidipes. This is explained by documented differences in the resting behaviour of the two species which shows that G. m. morsitans normally rests for longer periods on the surface of an untreated black target than does G. pallidipes. Other experiments showed that the presence of sebum elicited a probing response in G. m. morsitans and G. pallidipes. The results are discussed with reference to the possible use of host sebum to improve trap catches in the field.     

Two Tsetse fly species, Glossina palpalis gambiensis and Glossina morsitans morsitans, carry genetically distinct populations of the secondary symbiont Sodalis glossinidius

Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.     

Comparative study on the susceptibility of different laboratory strains of Glossina species to Trypanosoma simiae

Abstract. Teneral tsetse of four Glossina species from laboratory-reared colonies were fed on four Large White pigs infected with three different stocks of Trypanosoma simiae isolated in Coast Province, Kenya. Thereafter the tsetse were maintained on goats and dissected on day 28 to determine the trypanosome infection rates. Glossina brevipalpis was as susceptible as G.pallidipes whilst G.palpalis gambiensis was not susceptible to T.simiae CP 11 a stock causing acute infection, which was isolated from a wild G.austeni. Glossina brevipalpis was as susceptible as G.pallidipes to another stock causing acute infection, T.simiae CP 813 isolated from a wild G.pallidipes. Glossina morsitans centralis was also as susceptible as G.brevipalpis and G.pallidipes whilst G.p.gambiensis was not susceptible to this T.simiae stock. Glossina m.centralis showed very low susceptibility to a stock causing chronic infection, T.simiae CP 1896 isolated from a bushpig, whilst G.brevipalpis, G.p.gambiensis and G.pallidipes could not be infected by this T.simiae stock. Male Glossina were generally more susceptible than females to the three T.simiae stocks.     

Comparison between pars intercerebralis neurosecretory cells of Glossina and those of other higher Diptera

Three types of the A-neurosecretory cells (NSCs) were found in the pars intercerebralis (PI) of the adult brain of four species of tsetse flies (Glossina palpalis, G. pallidipes, G. austeni and G. morsitans). The typical formula of their composition is: 8 A1-, 14 A2- and 4 A3-cells. After permanganate oxidation, the neurosecretory materials (NSMs) of Al- and A3-cells are rich in strong acid groups. The A2-NSM contains both strong and weak acid groups after this treatment; besides, it retains a weak affinity to acid dyes. In addition to the A-cells, the PI of Glossina species seems to include a number of NSCs of the type B.
The data obtained indicate a great similarity of the NSC composition in the PI of Glossina and other related higher dipterans.
In addition to the NSCs, giant neurons and particular cells with large cytoplasmic inclusions were also found in the PI of Glossina. The cells with large cytoplasmic inclusions correspond apparently to vacuolated cells previously observed in the brain of some flies. It is suggested that the considerable complication in reproductive biology which took place in tsetse flies had no effect on the composition of the NSCs in their PI when compared to that in related species with more ordinary cycles of reproduction.     

Predicted distribution and movement of Glossina palpalis palpalis (Diptera: Glossinidae) in the wet and dry seasons in the Kogo trypanosomiasis focus (Equatorial Guinea).

The aim of this study was to predict the distribution and movement of populations of the tsetse fly, Glossina palpalis palpalis (Diptera: Glossinidae), in the wet and dry seasons and to analyze the impact of the use of mono-pyramidal traps on fly populations in the Kogo focus in 2004 and 2005. Three Glossina species are present in Kogo: Glossina palpalis palpalis, major HAT vector in West-Central Africa, Glossina caliginea, and Glossina tabaniformis. The apparent density (AD) of G. p. palpalis clearly fell from 1.23 tsetse/trap/day in July 2004 to 0.27 in December 2005. A significant reduction in the mean AD for this species was noted between seasons and years. The diversity of Glossina species was relatively low at all the sampling points; G. p. palpalis clearly predominated over the other species and significantly dropped as a consequence of control activities. The predictive models generated for the seasonal AD showed notable differences not only in the density but in the distribution of the G. p. palpalis population between the rainy and dry season. The mono-pyramidal traps have proven to be an effective instrument for reducing the density of the tsetse fly populations, although given that the Kogo trypanosomiasis focus extends from the southern Equatorial Guinea to northern Gabon, interventions need to be planned on a larger scale, involving both countries, to guarantee the long-term success of control.     

Circadian activity patterns in three species of tsetse fly: Glossina palpalis, austeni and morsitans

ABSTRACT. Observations on the nature and control of spontaneous flight activity in tsetse flies have so far been made only on G. m. morsitans Westw. This paper reports preliminary observations of the same kind on two other species: G. p. palpalis (R.-D.) and G. austeni Newst. The flight pattern of all three species consisted of short bursts of activity (usually lasting <1 min) separated by long intervals of rest, changes in activity level occurring exclusively as changes in the frequency of these flight bursts. In constant conditions in LD 12:12, G. palpalis and G. austeni were almost totally diurnal, with strongly predominant afternoon activity peaks, but a slight tendency to decrease activity around noon as occurs to a much greater extent in G. morsitans. This rhythm persisted in constant darkness (DD), but whereas in most G. morsitans only the morning peak was retained in DD, in G. palpalis and G. austeni it was only the afternoon peak. The loss of the afternoon peak in G. morsitans may be due to the very low activity levels in DD. Food deprivation led to increased activity in all three species, most markedly so in G. palpalis and G. austeni , in which the amount of activity per day increased c. ten-fold over three days' post-emergence starvation.     

The effects of temperature, body mass and feeding on metabolic rate in the tsetse fly Glossina morsitans centralis

Abstract.  Metabolic rate variation with temperature, body mass, gender and feeding status is documented for Glossina morsitans centralis . Metabolic rate [mean ± SE; VCO₂= 19.78 ± 3.11 μL CO₂ h⁻¹ in males (mean mass = 22.72 ± 1.41 mg) and 27.34 ± 3.86 μL CO₂ h⁻¹ in females (mean mass = 29.28 ± 1.96 mg) at 24 °C in fasted individuals] is strongly influenced by temperature, body mass and feeding status, but not by gender once the effects of body mass have been accounted for. A significant interaction between gender and feeding status is seen, similar to patterns of metabolic rate variation documented in Glossina morsitans morsitans . Synthesis of metabolic rate-temperature relationships in G. m. centralis , G. m. morsitans and Glossina pallidipes indicate that biting frequency as well as mortality risks associated with foraging will probably increase with temperature as a consequence of increasing metabolic demands, although there is little evidence for variation among species at present. Furthermore, metabolic rate–body mass relationships appear to be similarly invariant among these species. These data provide important physiological information for bottom-up modelling of tsetse fly population dynamics.     

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.     

Prevalence of trypanosomes,salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa

Background

Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal.

Results

The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected.

Conclusion

The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.