Evidence for a discrete evolutionary lineage within Equatorial Guinea suggests that the tsetse fly Glossina palpalis palpalis exists as a species complex

Tsetse flies of the palpalis group are major vectors of Human African Trypanosomiasis in Africa. Accurate knowledge of species identity is essential for vector control. Here, we combine ribosomal internal transcribed spacer 1 ( ITS1 ), mitochondrial Cytochrome Oxidase 1 ( COI ) and microsatellites to determine the population structure and phylogenetic relations of Glossina p. palpalis in Equatorial Guinea. CO1 sequence data suggest that G. p. palpalis in Equatorial Guinea is a distinct subspecies from previously described G. p. palpalis in West Africa and Democratic Republic of Congo. Glossina p. palpalis in Equatorial Guinea and DRC share a common ancestor which diverged from West African G. p. palpalis around 1.9 Ma. Previous ITS1 length polymorphism data suggested the possible presence of hybrids in Equatorial Guinea. However, ITS1 showed incomplete lineage sorting compared with clearly defined COI groups, and data from 12 unlinked microsatellites provided no evidence of hybridization. Microsatellite data indicated moderate but significant differentiation between the populations analysed (Rio Campo, Mbini and Kogo). Moreover, unlike previous studies of G. p. palpalis , there was no evidence for heterozygote deficiency, presence of migrants or cryptic population structure. Variance effective population size at Rio Campo was estimated at 501–731 assuming eight generations per year. This study of the population genetics of G. p. palpalis in central Africa provides the first estimate of genetic differentiation between geographically separated G. p. palpalis populations.     

Geographical distribution of Glossina palpalis gambiensis and G.p.palpalis in Liberia

The two subspecies of Glossina palpalis (Robineau-Desvoidy) occurring in Liberia could be reliably separated morphometrically by measuring the width of the terminal dilatations of the male inferior claspers. Subspecies differentiation of female flies was less conclusive. Identification of flies from fifty-four sites revealed that most of Liberia lies in the belt of Glossina palpalis palpalis. However, pure and substantial populations of G.p.gambiensis Vanderplank occur north of 8 degrees 20'N in the north-west of Liberia.     

Intraspecific variability in natural populations of Glossina palpalis gambiensis from West Africa, revealed by genetic and morphometric analyses

Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) from West Africa (Senegal and Burkina Faso) were analysed for microsatellite DNA polymorphisms and size of the wings. In the overall sample a strong heterozygote deficiency was found at two polymorphic microsatellite loci. It led to a highly significant value of Fis (within-sample heterozygote deficit) in the western zone of Sideradougou area in Burkina Faso. Genetic differentiation was significant on a macrogeographic scale, i.e. between tsetse coming from Senegal and Burkina Faso. Wing measures also differed between these two countries; flies from Senegal appeared to be smaller. Microsatellite loci further allowed differentiation of populations of G. palpalis gambiensis trapped on the same hydrographic network a few kilometres apart. The results are interpreted as indicating that further investigations will allow the study of genetic variability of tsetse flies in relation to the dynamics of transmission of human and animal trypanosomoses.     

Estimation of the efficiency of the biconical trap for Glossina fuscipes fuscipes along the lake Victoria shore, Kenya

Prior to estimating the efficiency of the unbaited biconical trap for Glossina fuscipes fuscipes Newstead (Diptera:Glossinidae) the flying height of the insects was estimated using 1 m2 electrified nets placed at 0 and 4 m above the ground level. The degree of avoidance of these nets by the flies was determined by comparing catches in traps surrounded and those not surrounded by incomplete rings of nets. On the basis of the catches in traps surrounded by nets, the expected catches on both sides of the nets were computed and compared with the observed catches, to further estimate this degree of avoidance. About 48% of males and 35% of females were captured above one metre. An average of 61% of males and 40% of females appeared to avoid the nets. Between 18% and 40% fewer tsetse were caught in traps surrounded by an incomplete ring of nets of respectively 1 m and 2 m radius than in traps not surrounded. After corrections for net avoidance and flying height, it appeared important to determine the optimum radius for the incomplete ring of nets for a reliable efficiency estimate.     

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.     

Gorging response of Glossina palpalis palpalis to ATP analogues

ABSTRACT. The potencies of seventeen analogues of ATP as gorging inducers for Glossina palpalis palpalis were evaluated. The ranking for effective dose that induced half the flies to gorge (ED₅₀) was: A tetra P 5 ATP=2'd ATP ADP=2'd ADP > AMP-PNP > 3'd ATP 2'3'dd ATP > AMP-PCP > adenosine 5' triphosphate 2',3'dialdehyde AMP-CPP >> AMP. Females detect ATP and its analogues better than males. The ED₅₀ of ATP was 5 × 10^-7 M for teneral females and 1.5 × 10^-6 M for males. According to the potency order of the ATP analogues, the G.p.palpalis gustatory receptors recognizing ATP can be classified as P_2y purinoceptors.     

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.     

Effects of age, sex and hunger on the antennal olfactory sensitivity of tsetse flies

Abstract The effects of age on electroantennogram (EAG) responses were investigated in male and female Glossina morsitans morsitans and comparative studies on the effects of starvation and sex on the EAG in G.m. morsitans, G.austeni, G.tachinoides and G.fuscipes fuscipes were made. Stimuli were the vapours of l-octen-3-ol, 4-heptanone, 3-nonanone and acetone. EAG decreased with age in both sexes of G.m.morsitans , responses in 5-day-old flies already being significantly lower than those in 1-day-old flies. In G.m.morsitans and G.tachinoides , EAG responses of males were higher than those of females. In G.austeni and G.f.fuscipes , however, the reverse was found. With increasing starvation EAG sensitivity increased in both sexes of G.m.morsitans and G.tachinoides. In G.austeni and in G.f.fuscipes no clear effects of starvation were observed. Response spectra of the individual species to the four odour substances did not change with increasing hunger. It is concluded that receptor sensitivity may be modulated depending on the insect's needs. Possible mechanisms of regulation and significance of this modulation are discussed.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.     

Structure of some East African Glossina fuscipes fuscipes populations

Abstract.  Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) is the primary vector of human sleeping sickness in Kenya and Uganda. This is the first report on its population structure. A total of 688 nucleotides of mitochondrial ribosomal 16S2 and cytochrome oxidase I genes were sequenced. Twenty-one variants were scored in 79 flies from three geographically diverse natural populations. Four haplotypes were shared among populations, eight were private and nine were singletons. The mean haplotype and nucleotide diversities were 0.84 and 0.009, respectively. All populations were genetically differentiated and were at demographic equilibrium. In addition, a longstanding laboratory culture originating from the Central African Republic (CAR-lab) in 1986 (or before) was examined. Haplotype and nucleotide diversities in this culture were 0.95 and 0.012, respectively. None of its 27 haplotypes were shared with the East African populations. A first approximation of relative effective population sizes was Uganda > CAR-lab > Kenya. It was concluded that the structure of G. f. fuscipes populations in East Africa is localized.     

The interaction of taste and heat on the biting response of the tsetse fly Glossina fuscipes fuscipes

Abstract.Tsetse flies probe more on a heated surface with a trace of uric acid than on one without. Uric acid is one of the components of human sweat and it elicits spike responses from taste hairs on the flies' legs. In this paper it is examined how heat from the surface and taste interact to affect the biting behaviour of Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) over successive days of food deprivation. The biting behaviour consists of bouts of probing, both ambulatory and stationary, intercalated with short hops of flight. The number of bouts increases over successive days, whereas the average bout duration does not. Although uric acid alone could not induce the flies to probe, in combination with surface heat it affected the flies greatly. Average bout duration was two‐fold that on a heated surface not treated with uric acid. The frequency of bouts was not affected by uric acid. These experiments and auxiliary ones on mechanoreceptive input and odours lead to the insight that the factors which affect biting behaviour can be viewed as a hierarchy. The hierarchy extends from those that affect the onset of biting to those that affect its course.     

Polymorphic microsatellite markers for the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae), a vector of human African trypanosomiasis

Our understanding of Glossina fuscipes fuscipes, a major vector of sleeping sickness, has been severely constrained by a lack of genetic markers for mapping and population genetic studies. Here we present 10 newly developed microsatellite loci for this tsetse species. Heterozygosity levels in Moyo, an Ugandan population, averaged 0.57, with only two loci showing very low heterozygosity. Five loci carried more than six alleles. Together with five recently published microsatellite loci, this brings the number of available microsatellite loci for this species to 15. Their availability will greatly facilitate future studies on the genetics of this important human disease vector.     

Effects of vegetation and weather on trap catches of Glossina fuscipes fuscipes near Lake Victoria, Kenya

Glossina fuscipes fuscipes Newstead was sampled in isolated thickets and forest patches near Lake Victoria, Kenya using unbaited biconical traps, between March 1992 and June 1993. Traps set at 1 m from the forest edge caught 3.3 times as many males and 5 times as many females as those set inside or 10 m away. The corresponding figures at 1 m from the edge of thicket were about 1.43 and 1.64 times, respectively. Hourly catches of males and females were positively correlated with temperature, light intensity and host (monitor lizard) prevalence, and negatively correlated with relative humidity. Light intensity and temperature were the most important variables affecting the catches of each sex. The results are discussed in relation to control and monitoring of G. f. fuscipes using traps.     

The diurnal activity, movement and trypanosome infection rates of Glossina fuscipes fuscipes （Diptera： Glossinidae） in Buvuma Island, Lake Victoria, Uganda

The diurnal activity patterns, trypanosome infection rates and movement of Glossinafuscipesfuscipes （Diptera： Glossinidae） were investigated in Buvuma Island, Lake Victoria, Uganda. Hourly trapping of tsetse flies was undertaken to determine their activity rhythm while a capture-mark-release-recapture method was conducted to assess the movement and dispersal of tsetse flies between lakeshore, hinterland and further inland sites along a transected area. Dissection of tsetse flies was also undertaken to determine the trypanosome infection rates in salivary glands, proboscis and mid-gut. Results indicated a bimodal diurnal activity profile for G. f fuscipes on the Island, both on the lakeshore and in the hinterland. Movement and dispersal of G. f fuscipes tsetse flies occurred between lakeshore, hinterland and further inland sites with a greater tendency of flies to move to the lakeshore. Trypanosome infection rates of 4.32% for Trypasoma vivax and 1.15% for 7. congolense were found in G. f. fuscipes.     

New epidemiological features on animal trypanosomiasis by molecular analysis in the pastoral zone of Sideradougou, Burkina Faso

A multidisciplinary work was undertaken in the agropastoral zone of Sidéradougou, Burkina Faso to try to elucidate the key factors determining the presence of tsetse flies. In this study the PCR was used to characterize trypanosomes infecting the vector ( Glossina tachinoides and Glossina palpalis gambiensis ) and the host, i.e. cattle. A 2-year survey involved dissecting 2211 tsetse of the two Glossina species. A total of 298 parasitologically infected tsetse were analysed by PCR. Trypanosoma vivax was the most frequently identified trypanosome followed by the savannah type of T. congolense and, to a lesser extent, the riverine forest type of T. congolense , and by T. brucei . No cases of T. simiae were found. From the 107 identified infections in cattle, the taxa were the same, but T. congolense savannah type was more frequent, whereas T. vivax and T. congolense riverine forest types were found less frequently. A correlation was found between midgut infection rates of tsetse, nonidentified infections and reptile bloodmeals. These rates were higher in G.p. gambiensis , and in the western part of the study area. T. vivax infections were related to cattle bloodmeals, and were more frequent in G. tachinoides and in the eastern study area. The PCR results combined with bloodmeal analysis helped us to establish the relationships between the vector and the host, to assess the trypanosome challenge in the two parts of the area, to elucidate the differences between the two types of T. congolense , and to suspect that most midgut infections were originating from reptilian trypanosomes.     

Trypanosomiasis in Kinshasa: distribution of the vector, Glossina fuscipes quanzensis, and risk of transmission in the peri-urban area

Abstract. Because human and animal cases of African trypanosomiasis have been reported in and around the city of Kinshasa for a long time, the likelihood of local transmission was examined. A georeferenced image of the city was produced, based on a satellite image (SPOT 4). Urban, peri-urban and rural areas were delineated. All recent data on captures of Glossina fuscipes quanzensis Pires (Diptera: Glossinidae) between 1999 and 2004, as well as epidemiological data on a 1999 outbreak of human trypanosomiasis by Trypanosoma brucei gambiense in the Kisenso District, were entered in a geographical information system (GIS). Tsetse flies were mainly found along some of the major rivers in the rural and peri-urban area of Kinshasa. Unsupervised classification of the satellite image allowed identification of riverine habitats suitable for tsetse flies and indicated sites where further entomological surveys were needed. The study produced strong indications that local transmission of human trypanosomiasis had occurred in the recent past in the peri-urban zone of Kinshasa.     

New data on parasitization of Glossina palpalis palpalis (Diptera: Glossinidae) by Hexamermis glossinae (Nematoda: Mermithidae) in a forested area of Ivory Coast

Observations on the parasitism of Glossina palpalis palpalis by Hexamermis glossinae were carried out over a period of one year by catching flies at Abengourou, Aboisso and Daloa (forested area of Ivory Coast). No parasite is observed out of 2,168 Glossina palpalis palpalis caught in Abengourou and 9,732 in Aboisso. At Daloa, dissections of 7,341 Glossina reveal 1.75% parasited flies. All the worms were located in the abdominal cavity, loosely intertwined with the internal organ. Males were more infested than females (2.68% versus 1.26%). The parasites were more abundant among the nulliparous (2.30%) than the young parous (1.19%) and than the old parous (0.52%). The majority of infected flies were caught at the beginning of the rainy season (5.17%) and few in the dry season (0.23%). The low parasitic infection rate observed here indicates a minimal effect on the population dynamics of the vector of sleeping sickness in Ivory Coast.     

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.     

Prospects of a fungus-contamination device for the control of tsetse fly Glossina fuscipes fuscipes

The prospects of the fungus Metarhizium anisopliae (Metsch.) Sorok. applied in contamination devices (Cds) to control tsetse fly Glossina fuscipes fuscipes Newstead was tested in a field experiment in Lake Victoria from 2 March 1999 to 31 August 2000. One hundred and sixty pyramidal traps mounted with Cds were deployed along the lakeshore and rivers on Mfangano Island. Contamination devices were loaded with 1.5-2.0 g of dry conidia/Cd. On the second island, Nzenze Island, four pyramidal traps fitted with plastic bags were deployed and served as the conventional 'trap and kill' population suppression method. A third island, Ngodhe Island, remained untreated and served as a control. Cds were recharged monthly with fresh conidia; plastic bags were also changed monthly. The apparent changes in population density were monitored weekly using biconical traps set at random on the three islands. To assess the incidence of M. anisopliae in tsetse flies on Mfangano Island, flies captured during monitoring were maintained in the laboratory and their mortality recorded. Fly population was reduced to 82.4 and 95.8% relative to untreated control on Mfangano and Nzenze islands, respectively, during the experimental period. Compared to the fungus-treated island, the number of flies caught in monitoring traps increased considerably in 'trap kill' treatment at 5 months after the treatments were removed. The incidence of M. anisopliae in fly populations was low during the 12 weeks following the initiation of the experiment but increased afterward until termination of the treatment. M. anisopliae could still be recovered from fly populations at 3 months after termination of the treatment, although the incidence was low. The results of this study have shown that application of M. anisopliae in a contamination device can suppress the population of G. fuscipes fuscipes comparable to the 'trap and kill' technology.