Tissue distribution and prevalence of Wolbachia infections in tsetse flies, Glossina spp

Tsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ-line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia-specific PCR-based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues.     

Starvation and desiccation in tsetse flies (Glossina)

Collections of tsetse flies were made from different regions and at different seasons, and their fat and water contents determined. The results showed that of the two potential causes of death, starvation is far more important than desiccation.

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Zusammenfassung In verschiedenen Gegenden und zu verschiedenen Jahreszeiten wurden Tsetsefliegen gesammelt und ihr Fett- und Wassergehalt bestimmt. Durch Extrapolation der Regressionen an Fett- und Wasserreserven konnten Schätzungen der nach Erschöpfung der Fettreserven übrigbleibenden Wasserreserven ermittelt werden, wie auch umgekehrt der übrigbleibenden Fettreserven nach Erschöpfung der Wasserreserven. Die Ergebnisse zeigten, dass von diesen zwei möglichen Todesursachen der Hunger viel wichtiger ist als das Vertrocknen. Dieses Ergebnis wurde durch eine Prüfung der Verteilung des Fett- und Wassergehalts bestätigt. Sie zeigte, dass sich eine beträchtliche Anzahl der Fliegen in der kritischen Zone des Fettgehalts oder sehr nahe daran befand, während nur ein geringer Bruchteil von ihnen einen Wassergehalt aufwies, der sich der kritischen Zone näherte.

     

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.     

Flying mate detection and chasing by tsetse flies (Glossina)

Abstract Male tsetse flies, probably Glossina morsitans morsitans Westw., were video-recorded in the field as they took off and chased other tsetse flies. Chasers responded (took off) to a target fly at a maximum distance of c. 55 cm, when it subtended c. 1.6^o to their eye (–1 foveal ommatidial subtense). Chased targets were always within this range (mean subtense at take-off = 3.2^o) and approaching the chaser. The most significant difference between chased and non-chased targets was in the rate of approach of the target fly in terms of the increase in its image size immediately before the chaser took off ( 21^o s⁻¹), especially as its relative increase (690% s^-1 P< 0.005). No feature of the target's translational velocity, nor any relationship between that and the image size approached this level of significance. Chasers seemed to 'slipstream' their target at c. 20 cm directly behind it, perhaps suggesting target identification by speed matching. Chases were apparently abandoned when the target image shrank from covering at least two of the chaser's foveal ommatidia to covering only one. Parallax-free measurements of flight speeds indicated a preferred, stable mean groundspeed of 4.8±0.1 m s^_1 (SE), at a mean wing-beat frequency of 209±3 Hz.     

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.     

The mating behaviour of tsetse flies (Glossina): a review

Abstract. The mode of reproduction of tsetse flies, by adenotrophic viviparity, is unusual among the Diptera and is associated with many unique aspects of the tsetse's mating system. Tsetse exist at relatively low densities in the environment but a combination of olfactory and visual stimuli brings males and virgin females together on or around host animals. The behavioural repertoire associated with mate location and identification, courtship and copulation is regulated by external physical and chemical stimuli as well as by internal physiological mechanisms. With a view to identifying stimuli that could be used to manipulate tsetse behaviour and exploited for control purposes, much progress has been made in recent years in elucidating the mating behaviour of tsetse and its regulatory mechanisms. This progress and the current state of understanding of tsetse mating behaviour is reviewed.     

Regulation of last reproduction steps in tsetse flies (Glossina: Diptera)

Summary

The regulation of ovulation and parturition, in the viviparous tsetse flies, constitutes a very precisely adjusted mechanism, Indeed the process of vitellogenesis takes places, in one oocyte only, during the incubation period when the larva is nourished in utero on milk produced by the mother. Neuropeptides, biogenic amines, neurotransmitters as well as ecdysone, and 20-hydroxyecdysone and also nervous outputs regulate ovulation and parturition. A model is proposed for the mechanisms controlling these processes.     

Tracking the feeding patterns of tsetse flies (Glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source.     

Behaviour of tsetse flies (Glossina) in host odour plumes in the field

ABSTRACT. In Zimbabwe, studies were made of the responses of Glossina pallidipes Austen and G.morsitans morsitans Westwood to artificial host odour using an incomplete ring of electrocuting nets. In a plume of synthetic host odour tsetse flew generally upwind, with 50–60% flying within 35^o of due upwind. More than 80% of tsetse flew at < 50 cm above ground level. Upon losing contact with odour they executed a reverse turn within about 2 m, and upon regaining contact they turned upwind. There were no clear differences in the responses of G.m.morsitans and G.pallidipes. Using electrocuting nets lying horizontally on the ground it was found that tsetse landed in the vicinity of the odour source, the propensity to land being greater for G.pallidipes than for G.m, morsitans , greater for immature than mature flies, and greater for males than females.     

Wind speed effects on odour source location by tsetse flies (Glossina)

Abstract. Tsetse flies (mainly Glossina pallidipes Aust.) were captured by various means at sources of artificial host odour in Zimbabwe and Kenya. Their rates of arrival and flight directions were compared with simultaneous data on the wind's speed and direction, on time-scales ranging from 1 s to 30 min. It was predicted that because increasing wind speed up to 1 m s^-1 straightens out the airflow (Brady et al. , 1989) it will straighten out odour plumes, make them easier to navigate, and should therefore increase the rate of arrival of flies at an odour source. In the event, the relationship proved to be more complex, with both positive and negative correlations of arrival rate on wind speed. It seems there is a bimodal relationship: odour source finding is positively related to increasing wind speed in weak winds up to ∼0.5 m s^-1 (presumably as the odour plume straightens out), but is negatively related to increasing wind speed in strong winds above ∼1.0 m s^-1 (presumably due to increasing turbulence breaking up the odour plume).     

Observations on the orientation of tsetse flies (Glossina pallidipes) to wind-borne odours

ABSTRACT. Observations of the upwind flight of Glossina pallidipes Austen near a source of host odour show that in the absence of a visual target the insects tend to overshoot the odour source in fast, low flight. There is no sign of the crosswind 'casting' flight which characterizes the behaviour of moths under similar circumstances, except that a 180 turn is executed to bring the tsetse flies back to the vicinity of the odour source in downwind flight. This may be followed by a second overshoot and another 180 turn before the insects alight within a metre or so of the source. The results indicate that the orientation of tsetse flies to host odour may involve a step-wise approach to the odour source, providing an opportunity for assessment of wind direction when the insects are at rest between successive bursts of flight.     

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.     

Flight responses of tsetse flies (Glossina) to octenol and acetone vapour in a wind-tunnel

Abstract. Female Glossina morsitans morsitans Westwood were video-recorded in a wind-tunnel as they entered, in crosswind flight, a broad plume of either octenol or acetone (two components of ox odour). Both odours produced upwind turning responses (in-flight anemotaxis) to a range of concentrations, with thresholds at around 10^-8mg1^-l for octenol and 10^-6mg1^-1 for acetone. Kinetic responses were unaffected by octenol at low concentrations, but flight speed was significantly reduced and sinuosity (^om^-1) and angular velocity (^os^-1) significantly increased by concentrations at or above those in ox breath; for acetone, these effects were apparent but inconsistently related to concentration. It is concluded that octenol and acetone vapour are used by tsetse flies to locate hosts by upwind anemotaxis, probably combined with kinetic responses. The behavioural basis for the 'repellency' of high octenol concentrations in the field is discussed in the context of the virtual loss of upwind anemotaxis to octenol at the highest concentration tested in the tunnel (30 × ox breath).     

Reciprocal regulation of fat content and flight activity in male tsetse flies (Glossina palpalis)

Abstract. To disentangle cause and effect in previously observed relationships between fat content and flight activity in male tsetse ( Glossina spp.), three groups of flies were fed at different intervals to raise their fat content to different levels before their flight activity was recorded. The greater the mean daily blood intake, the higher the fat content and the greater the subsequent spontaneous flight activity, thereby using up almost all of the fat reserves before the next blood meal. It is proposed that although male flies would benefit from maximum food intake to permit maximum flight associated with mate-seeking, they do not in fact feed as often as possible either in the field or the laboratory. This is explicable if energy acquisition is constrained by an additional mortality risk associated with feeding.     

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.      

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.     

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.

     

The causes of mating abstention in male tsetse flies Glossina morsitans

ABSTRACT. The sexual response of a Glossina morsitans morsitans Westw. male towards a decoy consisting of a dead male dosed with synthetic sex pheromone, was depressed by the presence of the cuticular lipids of the decoy male. However, addition of a C24 paraffin, not normally found on or in the cuticle of male or female G.m.morsitans , to a decoy male from which cuticular lipids were removed had a similar effect. Addition of either the C24 paraffin or an extract of male cuticular lipids to decoy females which possess natural sex pheromone in their cuticles, again depressed the sexual responses of males towards the decoys. The n-hexane solvent used in the application of compounds to decoys, itself depressed responses towards natural pheromone on a dead female at volumes in excess of 100μl, presumably because it mixed the surface waxes which masked the pheromone. Occlusion experiments implied that sex pheromone receptors are present on both the tarsi and tibiae of the male. No evidence was found for the existence of a specific mating deterrent on or in the cuticle of male G.m. morsitans.     

Changing visual responsiveness in pregnant and larvipositing tsetse flies, Glossina morsitans

ABSTRACT. Direct observations on the spontaneous activity and visually-evoked responses of pregnant female Glossina morsitans morsitans Westw. were made hourly during the photophase over the 4 days around larviposition. Visual stimulation was by a 5-wide vertical black stripe moving laterally, 4 times min^-1 at c. 5 s^-1. The numbers of take-offs and the numbers of females taking off were recorded, both in response to the stimulus and spontaneously in its absence. The visual responsiveness paralled both the day-to-day changes and the altered circadian patterns of spontaneous activity reported previously (Brady & Gibson, 1983), being very low for the 48 h before parturition. It did not, however, rise in parallel with the increase in spontaneous activity during the last 3–4 h before parturition, and the proportion of flights oriented towards the moving stripe was also much reduced at that time. The significance of this changing responsiveness to slow movement is discussed, and its potential enhancement of any behavioural avoidance of aerosol, non-residual insecticide spraying during late pregnancy is noted.