High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.)

Background

Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control.

Results

In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species.

Conclusions

The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

     

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)     

Upwind flight of tsetse (Glossina spp.) in response to natural and synthetic host odour in the field

Abstract. In Zimbabwe, studies were made of the flight responses of tsetse ( Glossina spp.) to synthetic and natural ox odour using arrangements of electric nets.Tsetse flying away from a target showed a significant upwind bias when a blend of carbon dioxide (2/1 min), acetone (500 mg/h), octenol (0.4 mg/h), 4-methylphenol (0.8 mg/h) and 3-n-propylphenol (0.1 ma) was dispensed 15 m upwind, with c. 35% flying upwind.Without carbon dioxide this percentage was significantly reduced to 15% which was not significantly different from that with no odour (8%).This pattern was not altered by reducing the doses of acetone, octenol and phenols by 10–100 times, to levels comparable to those produced by an ox.With natural ox odour or a synthetic equivalent of ox odour dispensed from a ventilated pit 8 m upwind of the target, c. 28% flew upwind.This was reduced significantly to 15% if carbon dioxide was removed.In studies using a 17 m line of nets arranged orthogonally across the prevailing wind line, c. 50% of the catch was caught on the downwind side in the absence of odour.This increased significantly to c.60% when acetone, octenol and phenols were dispensed 15 m upwind, with or without carbon dioxide.With a shorter line (9 m) or an incomplete one (16.5 m long with 5 times 1.5 m wide gaps along its length) there was no change in the proportion caught downwind.For all three lines, dispensing odour upwind increased the catch 2–5 times on both the up-and downwind sides of the nets.It is concluded that a stronger upwind response to host odour is elicited when carbon dioxide is present.It is suggested that in nature upwind flight is very imprecisely orientated, with tsetse making flights up and down an odour plume 'searching' for a host.     

A general model for mortality in adult tsetse (Glossina spp.)

Tsetse exhibit a U-shaped age-mortality curve, with high losses after eclosion and a well-marked ageing process, which is particularly dramatic in males. A three-parameter (k(1) -k(3) ) model for age-dependent adult instantaneous mortality rates was constructed using mark-recapture data for the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae). Mortality changed linearly with k(1) over all ages; k(2) affected only losses in roughly the first week of adult life, and k(3) controlled the ageing rate. Mortality pooled over age was twice as sensitive to changes in k(3) as in k(1) . Population growth rate was, however, similarly affected by these two parameters, reflecting the disproportionate effect of k(3) on mortality in the oldest flies that contribute least to the growth rate. Pooled-age mortality and growth rate were insensitive to changes in k(2) . The same model also provided good fits to data for laboratory colonies of female G. m. morsitans and Glossina austeni Newstead and should be applicable to all tsetse of both sexes. The new model for tsetse mortality should be incorporated into models of tsetse and trypanosome population dynamics; it will also inform the estimation of adult female mortality from ovarian dissection data.     

Some advances in the trapping of tsetse (Glossina spp.) and other flies

Abstract. 1. Shortcomings in the methodology of testing mechanical traps for tsetse and other flies have been partically overcome by relating all trap efficiencies to that of electric trapping devices which have been shown, independently, to capture over 95% of tsetse colliding with them.
2. In Rhodesia the classical 'animal' type traps only caught a small percentage of tsetse which approached them. The addition of ox odour increased the number of tsetse visiting the trap but did not affect trap efficiency.
3. Changes in trap design have resulted in increases in trapping efficiency of up to 4–5-fold over classical designs.
4. The addition of large quantities of ox odour increased the efficiency of the most successful trap described here, as well as the absolute number of flies taken. When the odour of livestock of total mass 11.5 tonnes was used, over 2000 tsetse could be trapped in a 3 h period.
5. None of the traps described here was particularly suitable for tabanids but some were used to trap large numbers of biting muscids.
6. The implications for new methods of tsetse control are discussed.     

Neural coding in antennal olfactory cells of tsetse flies (Glossina spp.)

Spike trains from individual antennal olfactory cells of tsetse flies (Glossina spp.) obtained during steady-state conditions (spontaneous as well as during stimulation with 1-octen-3-ol) and dynamic stimulation with repetitive pulses of 1-octen-3-ol were investigated by studying the spike frequency and the temporal structure of the trains. In general, stimulation changes the intensity of the spike activity but leaves the underlying stochastic structure unaffected. This structure turns out to be a renewal process. The only independently varying parameter in this process is the mean interspike interval length, suggesting that olfactory cells of tsetse flies may transmit information via a frequency coding. In spike records with high firing rates, however, the stationary records had significant negative first- order serial correlation coefficients and were non-renewal. Some cells in this study were capable of precisely encoding the onset of the odour pulses at frequencies up to at least 3 Hz. Cells with a rapid return to pre-stimulus activity at the end of stimulation responded more adequately to pulsed stimuli than cells with a long increased spike frequency. While short-firing cells process information via a frequency code, long-firing cells responded with two distinctive phases: a phasic, non-renewal response and a tonic, renewal response which may function as a memory of previous stimulations.      

Diuresis in the tsetse fly Glossina austeni.

After taking a blood meal, the tsetse fly Glossina austeni excretes the excess water and salts of the meal in approximately 30 min. During this period a volume of fluid equivalent to 80% of the unfed weight of the fly passes through the haemolymph, whose composition nevertheless remains almost constant. The fluid excreted has a higher sodium and lower potassium concentration than the haemolymph, indicating that sodium may be the prime mover in urine formation in Glossina.     

Octopamine distribution in the tsetse fly Glossina morsitans

Tissues of Glossina morsitans were assayed for octopamine using an enzymatic technique. Octopamine was detected at the highest concentration in the brain (7.06-7.99 ng mg-1 tissue protein) and thoracic ganglion (10.9-13.89 ng mg-1 tissue protein). Octopamine was present in haemolymph at a concentration of 1.0-1.27 X 10(-7) M. This was not found to vary when insects were flown or mechanically stressed. Nervous tissue, flight muscle and haemolymph showed a significant ability to metabolize octopamine. The greatest enzyme activity was present in the haemolymph.     

Impact of Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) on a heterologous tsetse fly host,Glossina fuscipes fuscipes

Background

Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomosis, the causal agent of sleeping sickness in humans and nagana in animals. Glossina fuscipes fuscipes is one of the most important tsetse vectors of sleeping sickness, particularly in Central Africa. Due to the development of resistance of the trypanosomes to the commonly used trypanocidal drugs and the lack of effective vaccines, vector control approaches remain the most effective strategies for sustainable management of those diseases. The Sterile Insect Technique (SIT) is an effective, environment-friendly method for the management of tsetse flies in the context of area-wide integrated pest management programs (AW-IPM). This technique relies on the mass-production of the target insect, its sterilization with ionizing radiation and the release of sterile males in the target area where they will mate with wild females and induce sterility in the native population. It has been shown that Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) infection causes a decrease in fecundity and fertility hampering the maintenance of colonies of the tsetse fly G. pallidipes. This virus has also been detected in different species of tsetse files. In this study, we evaluated the impact of GpSGHV on the performance of a colony of the heterologous host G. f. fuscipes, including the flies’ productivity, mortality, survival, flight propensity and mating ability and insemination rates.

Results

Even though GpSGHV infection did not induce SGH symptoms, it significantly reduced all examined parameters, except adult flight propensity and insemination rate.

Conclusion

These results emphasize the important role of GpSGHV management strategy in the maintenance of G. f. fuscipes colonies and the urgent need to implement measures to avoid virus infection, to ensure the optimal mass production of this tsetse species for use in AW-IPM programs with an SIT component.

     

Female meiosis in two species of tsetse fly (Genus Glossina)

Supernumerary chromosomes form bivalents during female meiosis in the tsetse fly and it is suggested that this is a control mechanism for their retention in the population. Chiasmata regularly form in the autosome and X chromosome bivalents.     

Influence of diet composition on feeding and water excretion by the tsetse fly, Glossina morsitans

Adult Glossina morsitans fed on aqueous salt solutions containing phagostimulant ATP in an in vitro feeding system gave an optimal feeding response only over a narrow pH range equivalent to that of vertebrate blood. There was much less discrimination on the basis of molar concentration.The rate and extent of water excretion by the fly was found to depend on the concentration of Na⁺ ions in the food medium: an active transport mechanism is indicated which enables water to pass from the meal through the anterior midgut wall and into the haemocoele. A favourable osmotic gradient assisted water transport in the presence of Na⁺ ions: the system could not operate efficiently in the presence of Na⁺ ions if the osmotic pressure of the food medium was higher than that of vertebrate blood, nor could it operate efficiently in any solution lacking Na⁺ ions.Normal transfer of a meal from the crop to the anterior midgut occurred only when the food medium was isotonic with vertebrate blood or in the presence of Na⁺ ions if hypotonic. Normal transfer of isotonic solutions was prevented in the presence of excess K⁺ ions, and hypertonic solutions were not transferred normally even in the presence of Na⁺ ions. Thus the rate of water excretion was reduced.Tsetse flies fed on blood in an in vitro feeding system excreted water at a significantly lower rate than flies fed on a living animal. Evidence suggests that this is due to a combined effect of changes in viscosity, effective ionic composition, and osmotic pressure, upon the normal rate and extent of food uptake and manipulation of the meal prior to digestion. The implications of this are discussed in terms of future developments of in vitro feeding techniques for haematophagous insects.     

Juvenile hormone mimics as effective sterilants for the tsetse fly Glossina morsitans morsitans

The development of puparia of Glossina morsitans morsitans Westwood was disrupted by topical applications of the juvenile hormone mimics S-methoprene (the resolved enantiomer of 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoic acid 1-methyl ester) (Zoecon), S21149 (propionaldoxime-0-4-phenoxyphenoxyethylether) (Sumitomo), or S31183 (2-[1-methyl-2-(4-phenoxyphenoxy)ethoxy]pyridine) (Sumitomo) dissolved in acetone. Puparia so treated during the first 4 days of life suffered developmental abnormalities, the severity of which were dose-dependent. Similarly, puparia produced by adult females treated with these compounds were abnormal. Dose-response data showed that effects were greatest with S31183 and least with S-methoprene. Abnormalities in the form of abdominal lesions and wing crumpling were typical of flies emerging from puparia produced by S-methoprene-treated females. However, arrested development at the red eye and pigmented seta stage within the puparium were typical of offspring of females treated with S21149 and S31183. A dose of 2 micrograms per female of S31183 was sufficient to prevent emergence of offspring produced for the rest of the life of the fly. The same dose resulted in partial recovery of females treated with S21149 some 18 days following treatment. Treatment with 2 micrograms S-methoprene did not suppress completely the production of normal offspring and recovery was complete some 27-35 days after treatment. Exposure of males to 20 micrograms S31183 did not impair their ability to inseminate females; transfer of material during copulation was sufficient to prevent the production of viable offspring by their mates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.