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.     

Genetics of two colonies of Glossina pallidipes originating from allopatric populations in Kenya

Abstract Two large colonies, originating from allopatric populations of Glossina pallidipes Austen, in the Shimba Hills and Nguruman, Kenya, which differ biologically and with respect to vectorial competence, were compared at fourteen enzyme loci using polyacrylamide gel electrophoresis. The colonies had similar levels of genetic diversity with approximately half of the loci being polymorphic, an average of 1.6-1.7 alleles per locus, and a mean heterozygosity per locus of approximately 18.4%. However, the colonies differed significantly in allele frequencies at the loci for phosphoglucomutase, glucose-6-phosphate dehydrogenase, xanthine oxidase, octanol dehydrogenase and phosphoglucose isomerase. The results were compared with earlier studies on this species and no evidence was found for selection of specific alleles during establishment or maintenance of colonies of G.pallidipes , nor were specific chromosomes, or marker genes, associated with the biological differences between the two colonies.     

New Polymorphic Microsatellites in Glossina pallidipes (Diptera: Glossinidae) and Their Cross-Amplification in Other Tsetse Fly Taxa

We report the development and characterization of three new microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae). Fifty-eight alleles were scored in 192 individuals representing six natural populations. Allelic diversity ranged from 9 to 28 alleles per locus (mean 19.3 +/- 5.5). Averaged across loci, observed heterozygosity was 0.581 +/- 0.209, and expected heterozygosity was 0.619 +/- 0.181. Cross-species amplifications of the G. pallidipes loci in other tsetse fly taxa are reported.     

Characterization of microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Glossina pallidipes is a vector of African trypanosomiasis. Here we characterize eight new polymorphic microsatellite loci in 288 G. pallidipes sampled from 12 Kenya populations. The number of alleles per locus ranged from four to 36 with a mean of 20.5 +/- 10.1. Expected single locus heterozygosities varied from 0.044 to 0.829. Heterozygosity averaged 0.616 +/- 0.246. No linkage disequilibrium was found. We also report results in eight other tsetse species estimated by using the primers developed in G. pallidipes. The primers worked best in G. swynnertoni and G. austeni and worst in G. m. morsitans and G. m. submorsitans.     

Development of a low-cost tsetse trap and odour baits for Glossina pallidipes and G.longipennis in Kenya

Experiments were carried out to improve the NG2B tsetse trap (Brightwell et al., 1987), baited with acetone and cow urine, for use by rural communities to control G.pallidipes Austen and G.longipennis Corti. Modifications included a lower dose rate of acetone, a new cage design and raising the trap about 15-20 cm. Research on different trap cone materials showed that the degree of light transmission of the netting, rather than its colour, was the crucial factor affecting the catch of G.pallidipes. Adding an additional metre of blue cloth to one side of the trap increased catches of females of both species by about 60%. Traps baited with synthetic phenols yielded similar numbers of G.pallidipes and significantly more G.longipennis than those baited with natural cow urine. The latter difference was not apparent when octenol was also used, so cow urine was retained as one of the odour baits in preference to the imported phenols. Although octenol increased catches of G.pallidipes by only about 30%, catches of G.longipennis were increased 2-4-fold, making it a very useful attractant for the latter species. The cost of the trap/odour-bait system was estimated to be US$8.5 per unit per annum. The economics of this method of tsetse control are discussed.     

Abundance and distribution of the tsetse flies, Glossina austeni and G. brevipalpis, in different habitats in South Africa

The distribution and abundance of Glossina austeni Newstead and Glossina brevipalpis Newstead (Diptera: Glossinidae) were studied in the three main vegetation types in Zululand, KwaZulu-Natal, South Africa. During a period of 12 months, a trap transect consisting of 38 H-traps traversing the three vegetation types was monitored. The Index of Apparent Abundance (IAA) for G. brevipalpis was high in indigenous forest and open grassland but lower in exotic plantations. Glossina austeni, on the other hand, was captured mainly in or adjacent to indigenous forest. The seasonal trend in the IAA did not differ between vegetation types. The findings on the distribution of G. brevipalpis are in contrast with the historic records. Historically, this species was considered to be restricted to areas with a dense overhead canopy and high relative humidity. The repercussions of these findings for the epidemiology of livestock trypanosomiasis and the control of tsetse in Zululand are discussed.     

Analysis of the mating activity of male tsetse flies Glossina m. morsitans and G. pallidipes in the laboratory

ABSTRACT. The responses of male Glossina morsitans morsitans West-wood and Glossina pallidipes Austen to freeze-killed females were examined in the laboratory. Analyses were performed using a specially designed, automated, computer-based, recording system. G. pallidipes were more active than G. m. morsitans , interacting with the female decoys twice as often. Interactions with the decoys divided broadly into short-stay (<60 s) and long-stay, full copulatory attempts. For G. m. morsitans 90% of interactions resulted in full copulatory attempts, the mean duration of which was >1 h. For G. pallidipes only 40% of interactions resulted in full copulatory attempts, the mean duration of which was 35 min. The initiation of interactions showed a clear V-shaped activity pattern in G. m. morsitans but in G. pallidipes only a morning peak was observed. In neither species was there a tendency for full copulatory responses to be initiated in any specific period of the diurnal activity pattern. The results indicate that the two species have very different mating systems, and represent an initial step in the quantification of these differences.     

A comparison of African buffalo, N''Dama and Boran cattle as reservoirs of Trypanosoma congolense for different Glossina species

Teneral Glossina morsitans centralis Machado were fed on the flanks of the African buffalo (Syncerus caffer Sparrman), N'Dama (Bos taurus L.) or Boran (Bos indicus L.) cattle infected with Trypanosoma congolense Broden. The infected tsetse were maintained on rabbits and on day 30 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean infection rates (% +/- SE) in the midgut of tsetse fed on buffalo, N'Damas and Borans were 23.5 +/- 3.3, 31.6 +/- 2.7 and 33.7 +/- 4.6, respectively. The differences were not significant. However, the mean mature infection rate in tsetse fed on the buffalo (13.2 +/- 2.1%) was significantly lower compared to the rates in tsetse fed on the N'Dama (20.4 +/- 1.4) or the Boran cattle (21.4 +/- 1.1). When groups of teneral G.m.centralis, G.pallidipes Austen, G.p.gambiensis Vanderplank, G.f.fuscipes Newstead, G.brevipalpis Newstead and G.longipennis Corti were fed simultaneously on either an infected buffalo, an N'Dama or a Boran steer, the mature infection rates ranged from 0 to 16.1%. Irrespective of the host species used, the T.congolense infection rate was highest in G.m.centralis, lowest in the palpalis and fusca group tsetse, with G.pallidipes being intermediate. Nevertheless, the trypanoresistant African buffalo and N'Dama may serve as reservoirs of T.congolense as can trypanosusceptible Boran cattle.     

Selective use of odour-baited, insecticide-treated targets to control tsetse flies Glossina austeni and G. brevipalpis in South Africa

The effectiveness of odour-baited targets treated with 0.8% deltamethrin in controlling Glossina austeni Newstead and G. brevipalpis Newstead (Diptera: Glossinidae) was evaluated in Zululand, South Africa. Targets were initially deployed in the three habitat types (grassland, woodland and forest) of two adjacent areas at a density of four targets per km(2). One area functioned as the treatment block (c. 35 km(2)) and included the focus of the target deployment, and the second area functioned as a barrier block (c. 40 km(2)) against tsetse fly re-invasion from the untreated area to the south. After 8 months, targets were removed from open grassland in both areas and target density in wooded habitats and sand forest was increased to eight per km(2). Twelve months later, all targets were removed from the barrier block and used to increase target density in the wooded and sand forest habitats of the treatment block to 12 per km(2). This target density was maintained for 14 months. In the treatment area, a 99% reduction in G. austeni females occurred after 13 months at a target density of eight per km(2) in wooded habitat; this was maintained for 22 months. Reduction in G. brevipalpis was less marked. The relatively poor reduction in G. brevipalpis is attributed to the high mobility of this species and its distribution throughout less wooded and more open habitats.     

Activation of three species of tsetse (Glossina spp.) in response to host derived stimuli

Recordings were made of the activation of hungry Glossina morsitans morsitans Westwood, G. pallidipes Austen, and G. austeni Newstead in response to odours from ox breath and ox urine, and a moving visual stimulus, in a wind tunnel. The spontaneous activity of G.m.morsitans was very low (less than 4% of males and 2% of females active per min during control periods). That of G.austeni and G.pallidipes was in the region of 20% except for G.pallidipes females when in excess of 40% were active during control periods. Addition of ox urine odours to the airstream had no effect on activity in any of the species investigated but addition of ox breath odours to the airstream significantly increased activity of G.pallidipes and of G.m.morsitans, although for the latter only approximately 12% of flies were active. For G.austeni the addition of ox breath odours resulted in a significant increase in activity of females but not of males. The moving visual stimulus resulted in a significant increase in the activity of both sexes of G.austeni and G.m.morsitans but no change in the activity of G.pallidipes. The low level of spontaneous activity and the low response to ox breath odours in a strain of G.m.morsitans maintained in the laboratory since 1969 was compared with a new colony of this species which originated from puparia collected in Zimbabwe in 1991. No differences in either spontaneous activity or the response to ox breath odour was recorded, but females from the new colony were significantly more responsive to a moving visual stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses of tsetse flies, Glossina morsitans morsitans and Glossina pallidipes, to baits of various size

Recent studies of Palpalis group tsetse [Glossina fuscipes fuscipes (Diptera: Glossinidae) in Kenya] suggest that small (0.25 × 0.25 m) insecticide-treated targets will be more cost-effective than the larger (≥1.0 × 1.0 m) designs currently used to control tsetse. Studies were undertaken in Zimbabwe to assess whether small targets are also more cost-effective for the Morsitans group tsetse, Glossina morsitans morsitans and Glossina pallidipes. Numbers of tsetse contacting targets of 0.25 × 0.25 m or 1.0 × 1.0 m, respectively, were estimated using arrangements of electrocuting grids which killed or stunned tsetse as they contacted the target. Catches of G. pallidipes and G. m. morsitans at small (0.25 × 0.25 m) targets were, respectively, ～1% and ～6% of catches at large (1.0 × 1.0 m) targets. Hence, the tsetse killed per unit area of target was greater for the larger than the smaller target, suggesting that small targets are not cost-effective for use against Morsitans group species. The results suggest that there is a fundamental difference in the host-orientated behaviour of Morsitans and Palpalis group tsetse and that the former are more responsive to host odours, whereas the latter seem highly responsive to visual stimuli.     

Identification and properties of microsatellite markers in tsetse flies Glossina morsitans sensu lato (Diptera: Glossinidae)

Genomic libraries enriched for simple sequence repeats were constructed for Glossina morsitans morsitans, G. m. submorsitans, and G. m. centralis. Sixteen microsatellite markers were isolated from the libraries and evaluated on flies from natural G. m. morsitans populations and other Glossina species in the Morsitans and Palpalis species groups. The primers amplified appropriate sized DNA fragments in the Morsitans and Palpalis groups. In G. morsitans s.l., eight of 12 dinucleotide repeats and four of 12 trinucleotide repeats were polymorphic. The polymorphic loci showed a mean 7.5 +/- 4.8 alleles per locus and their mean heterozygosity was 55.8 +/- 7.7%.     

Effect of puparia incubation temperature: increased infection rates of Trypanosoma congolense in Glossina morsitans centralis, G.fuscipes fuscipes and G.brevipalpis

Puparia of Glossina morsitans centralis (Machado), G.fuscipes fuscipes (Newstead) and G.brevipalpis (Newstead) were incubated at 25 +/- 1 degrees C, 28 +/- 1:25 +/- 1 degrees C, day:night or 29 +/- 1 degrees C throughout the puparial period, and maintained at 70-80% relative humidity. Puparial mortality was higher at 29 than at 25 degrees C (optimum temperature) in all three species, particularly in G.f.fuscipes and G.brevipalpis. Adults of G.m.centralis from puparia incubated at 29 degrees C, and those of this subspecies, G.f.fuscipes and G.brevipalpis from puparia incubated at 28:25 degrees C, day:night or 25 degrees C throughout, were infected as tenerals (27 h old) by feeding them at the same time on goats infected with Trypanosoma congolense (Broden) IL 1180 after the parasites were detected in the wet blood film. Infection rates on day 25 post-infected feed were higher in G.m.centralis from puparia incubated at 29 degrees C and in adults of the three different tsetse species from puparia incubated at 28:25 degrees C, day:night, than in those from puparia incubated at 25 degrees C. However, in G.f.fuscipes the labral and hypopharyngeal infection rates were not significantly different from those of the tsetse produced by puparia kept at 25 degrees C.     

Genetic Variation at Structural Loci in the Glossina morsitans Species Group

Gene diversity was investigated in four taxa of tsetse flies (Diptera: Glossinidae) including Glossina morsitans morsitans, G. m. centralis, G. swynnertoni, and G. pallidipes. Histochemical tests were performed for 35–46 isozymes. Polymorphic loci were 20% in G. morsitans morsitans, 32% in G. m. centralis, 17.6% in G. swynnertoni, and 26% in G. pallidipes. Mean heterozygosities among all loci were 6.6% in G. morsitans morsitans, 6.0% in G. m. centralis, 7.1% in G. swynnertoni, and 6.8% in G. pallidipes. Allozyme gene diversities were considerably less than those reported for many Diptera. The low gene diversities are probably related to small effective population sizes.     

Tsetse mating behaviour: effects of age and hunger in Glossina morsitans morsitans and G.pallidipes

ABSTRACT. The effects of age and hunger on the responses of male Glossina morsitans morsitans Westwood and G.pallidipes Austen to freeze-killed female decoys, were examined in the laboratory. In both species, activity, estimated as the total number of interactions between males and decoys, increased with both age and hunger. Interactions were divided into short-stay (<60 s) and long-stay, full copulatory responses. In both species, young, unfed males were significantly less likely to attempt to copulate with a decoy after encounter than were fed males. Among fed males the proportion of interactions that proceeded to full copulatory attempts did not change with increasing age, but decreased consistently with increasing hunger. At all ages and hunger levels, G.pallidipes were more active than G.m.morsitans. However, after encountering a decoy, G.pallidipes were less likely to attempt to copulate than G.m.morsitans. In both species the duration of copulatory attempts did not change with age, but declined with increasing hunger. Copulatory attempts by G.pallidipes were significantly shorter than those of G.m.morsitans. The results are discussed in relation to the behaviour of tsetse in response to control devices such as traps and targets.     

Microgeographical breeding structure of the tsetse fly, Glossina pallidipes in south-western Kenya

The origins of extant Glossina pallidipes Austen (Diptera: Glossinidae) populations in the ecologically well-studied Lambwe and Nguruman valleys in Kenya are controversial because populations have recovered after seemingly effective attempts to achieve high levels of control. The microgeographical breeding structure of the tsetse fly, G. pallidipes, was investigated by analysing spatial and temporal variation at eight microsatellite loci to test hypotheses about endemism and immigration. Samples were obtained at seasonal intervals from trap sites separated by 200 m to 14 km and arranged into blocks. G. pallidipes populations nearest to Lambwe and Nguruman also were sampled. Spatial analysis indicated that genetic differentiation by genetic drift was much less among trapping sites within Lambwe and Nguruman (F(ST) < or = 0.049) than between them (F(ST) = 0.232). F(ST) between Serengeti and Nguruman was 0.16 and F(ST) between Kodera Forest and Lambwe was 0.15. The genetic variance in G. pallidipes explained by dry and wet seasons (0.33%) was about one-fifth the variance among collection dates (1.6%), thereby indicating reasonable temporal stability of genetic variation. Gene frequencies in Kodera and Serengeti differed greatly from Lambwe and Nguruman, thereby falsifying the hypothesis that Lambwe and Nguruman were repopulated by immigrants. Harmonic mean effective (= breeding) population sizes were 180 in Lambwe and 551 in Nguruman. The genetic data suggest that G. pallidipes in Lambwe and Nguruman have been endemic for long intervals.     

The role of cattle as hosts of Glossina longipennisat Galana Ranch, south-eastern Kenya

Abstract. Glossina longipennis were recorded visiting and engorging on cattle in an enclosure and on a single ox in a crush using transparent electrocuting nets in an incomplete ring. Of the total flies caught, 3–6% of males and 5–6% of females in the total catches were engorged (a feeding success rate of up to 16.6% and 12.6%, respectively, depending on assumptions made about the proportion which had an opportunity to feed). Direct observation of tsetse from an observation pit showed 57% landing on the front legs, 13% on the hind legs, and 11 % on the belly of the host. The largest number of bloodmeals was taken from the front legs, although only 14% of landings there terminated in feeding; a higher proportion of the flies alighting on the hind legs and flank succeeded in feeding (28% and 21% respectively). Glossina longipennis were attracted to targets baited with ox odour from an underground pit in a dose-dependent manner. Odour of humans was much less attractive to G.longipennis than that of oxen (for equivalent biomass). Analysis of bloodmeal samples from tsetse caught in two sites on die ranch showed that G.longipennis preferentially feeds on suids, bovids and hippopotamus.     

A comparison of susceptibility to stocks of Trypanosoma simiae of Glossina pallidipes originating from allopatric populations in Kenya

Abstract. A colony of Glossina pallidipes which originated from Nguruman, Rift Valley Province, Kenya, was significantly more susceptible than a colony of the same species which originated from Shimba Hills, Coast Province, Kenya, to infection with a stock of Trypanosoma simiae CP 11 isolated from wild G. austeni in Coast Province, Kenya, irrespective of whether pigs or goats were used as infecting hosts. Male G. pallidipes from both the colonies were more susceptible than females to this T. simiae stock. Similarly, a G. pallidipes colony of Nguruman origin was significantly more susceptible than the colony of Shimba Hills origin to infection with another stock of T. simiae CP 813 isolated from wild G. pallidipes in Coast Province, Kenya, again irrespective of whether pigs or goats were used as infecting hosts. The susceptibility of the sexes of G. pallidipes from both the colonies to T. simiae CP 813 did not differ significantly when pigs were used as infecting hosts, but male G. pallidipes from both the colonies were significantly more susceptible than female tsetse to this T. simiae stock when goats were used as infecting hosts. Nevertheless, if the observed differences in susceptibility of the two G. pallidipes colonies reflect transmission of trypanosomes by the two allopatric populations of tsetse in the field, then the epidemiology of simiae- trypanosomiasis probably differs between these two areas of Kenya.     

Ovulation, insemination and mating in the tsetse fly Glossina pallidipes

ABSTRACT. Analyses of the ovulation, insemination and mating of female Glossina pallidipes Austen, were carried out in the field and laboratory. In unmated females, ovulation was delayed until at least the fifteenth day of adult life. The predictable pattern of growth and development of eggs was used to age nulliparous females to the nearest day in the field. Inseminated females first appeared at 5–6 days old and most females became inseminated at 8–10 days of age. There were no significant differences in the age at insemination in Zimbabwe or Kenya. Ovulation had occurred in the field by 10–11 days old. Mating therefore occurs at or just before ovulation. Females were able to mate at up to 12 days old and still produce a viable larva from their first egg. In laboratory flies originating from Uganda, factors which reduced and minimized the effects of disturbance, maximized insemination rate, so that under the best conditions the age at mating was identical to that found in the field. Nevertheless, the insemination rate of Fl generation Zimbabwe flies reared from wild-caught females was negligible. The differences in the laboratory mating behaviour of the Zimbabwe flies and the flies from the long-established colony of Uganda origin, are considered to be due to wild G. pallidipes being easily disturbed under laboratory conditions, whereas colony breeding rapidly selects for passivity. The concept of gross differences in G. pallidipes mating behaviour between geographic areas in thus rejected.     

Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Tsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST=0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST=0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.