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.     

The effect of ambient temperature on the flight performance of the mature male tsetse fly, Glossina morsitans

ABSTRACT. The wingbeat frequency at flight initiation in mature male G.morsitans West, rose approximately linearly from 213 Hz at 20°C to 263 Hz at 32°C. An increase of temperature to 36°C produced no further effect. The time course of frequency changes was affected in a complex manner by temperature, due probably to the interaction of a number of physico-chemical and behavioural processes with different temperature dependencies. Higher temperatures caused a more rapid overall decline in wingbeat frequency, consistent with the greater demand on the limited reserve of the only important flight substrate, proline. At 20°C, a considerable proportion of tsetse were relatively inactive, whilst a small proportion flew for longer than at any other temperature. At intermediate temperatures both extremes in flight duration decreased, but at 36°C 80% of tsetse flew for less than 90s. These observations can be explained on the basis of the interaction between the tendency of tsetse to be inactive at extremes of temperature, and the more rapid oxidation of reserves at higher temperatures.     

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B₆) as a cofactor. This vitamin can be synthesized by tsetse''s obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B₆ for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse''s reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B₆ metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B₆ in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B₆ production.     

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.     

Distribution and Density of Tsetse Flies (Glossinidae: Diptera) at the Game/People/Livestock Interface of the Nkhotakota Game Reserve Human Sleeping Sickness Focus in Malawi

In large parts sub-Saharan Africa, tsetse flies, the vectors of African human or animal trypanosomiasis, are, or will in the foreseeable future, be confined to protected areas such as game or national parks. Challenge of people and livestock is likely to occur at the game/livestock/people interface of such infested areas. Since tsetse control in protected areas is difficult, management of trypanosomiasis in people and/or livestock requires a good understanding of tsetse population dynamics along such interfaces. The Nkhotakota Game Reserve, an important focus of human trypanosomiasis in Malawi, is a tsetse-infested protected area surrounded by a virtually tsetse-free zone. The abundance of tsetse (Glossina morsitans morsitans) along the interface, within and outside the game reserve, was monitored over 15 months using epsilon traps. A land cover map described the vegetation surrounding the traps. Few flies were captured outside the reserve. Inside, the abundance of tsetse at the interface was low but increased away from the boundary. This uneven distribution of tsetse inside the reserve is attributed to the uneven distribution of wildlife, the main host of tsetse, being concentrated deeper inside the reserve. Challenge of people and livestock at the interface is thus expected to be low, and cases of trypanosomiasis are likely due to people and/or livestock entering the reserve. Effective control of trypanosomiasis in people and livestock could be achieved by increasing the awareness among people of dangers associated with entering the reserve.     

Flying to feed or flying to mate: gender differences in the flight activity of tsetse (Glossina palpalis)

Abstract. To test the prediction that female and male tsetse should differ in their behaviour and the partitioning of their energy budgets to maximize their respective reproductive outputs, we investigated experimentally the relationship between blood intake, fat content and flight activity in virgin and mated female flies, following the same procedure as in a previous study on males.Those flies whose fat content was raised to higher levels by being fed more frequently performed more flight activity, but all females showed very little activity until day 4 after their last blood meal, thereby using only a small fraction of their fat reserves.This contrasted markedly with the large amount of flight performed by males on day 3, resulting in the depletion of their fat reserves.The difference is interpreted with respect to females flying only to find food approximately once every 3 days (and larviposition sites approximately once every 9 days), compared with males flying to find as many mates as possible during the earlier part of the feeding cycle when their energy reserves are high and feeding is a low priority.     

Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

The flight performance of tsetse flies.

The four species of tsetse used are shown to be aerodynamically rather similar. The wingbeat frequency, aerodynamic lift, flight duration, and number of wingbeats per flight are seen to undergo large increases during adult maturation. Tsetse can produce unusually large aerodynamic lifts, although the flight performance declines very quickly during individual flights. The large lifts appear to be associated with the high percentage of muscle in the fly, and the rapid decline with the inability of the fly to replenish rapidly its proline reserves. Wing mutilation has been shown to increase the wingbeat frequency. The potentiation of the flight performance during maturation appears to be causally linked to increases in the thoracic muscle, cuticle, and proline levels. The results presented here are at variance with those of an earlier study on the physics of flight in tsetse.     

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.     

The nutritional state of male tsetse flies,Glossina pallidipes,at the time of feeding

Abstract. The feeding intervals of tsetse flies have been estimated from the nutritional state of flies caught in traps. However, such estimates have been disputed on the grounds that traps catch a biased, hungry sample of the flies which are seeking hosts and will feed. In this paper we present data on the nutritional state of tsetse flies caught approaching and feeding on oxen. Individual oxen were surrounded with an incomplete ring of electric nets which caught Glossina pallidipes Austen that were approaching, departing unfed and departing fed from an ox. Non-teneral males caught in this way were analysed for their fat and haematin contents. The feeding interval was estimated from a comparison of the frequency distributions of the pre- and post-feed haematin contents of the flies which fed. The former was not measured directly, and was deduced from the frequency distributions of the haematin contents of the male flies caught approaching and departing unfed from the oxen, since it is assumed that the departing unfed and fed flies together form a sample of the approaching flies. There was no difference between the frequency distributions of haematin contents of flies caught approaching and departing unfed, and therefore the pre-feed haematin contents of the males which fed should have the same frequency distribution. Comparison of this distribution with that of the post-feed haematin contents of the males which fed indicated that the majority of male G.pallidipes were returning to feed after digesting on average 1.4 log haematin units of the previous bloodmeal. From data published elsewhere, this corresponds to a mean feeding interval of 42-60h. There was a strong, linear, negative relationship between the fat contents of males and their probability of taking a bloodmeal, suggesting that fat content is important in determining the feeding behaviour of tsetse flies.     

Proline transport by tsetse fly Glossina morsitans flight muscle mitochondria.

1. Proline accumulation by tsetse fly Glossina morsitans flight muscle mitochondria was studied in vitro by the swelling technique and direct measurement of (U-14C) proline. 2. Proline transport was inhibited by the uncharged liposoluble -SH reagent, N-ethylmaleimide but not by ionic reagent, mersalyl, suggesting that the -SH groups involved in the transport of proline are located in a hydrophobic part of the membrane or on the matrix side of the membrane. 3. The kinetic study of proline accumulation revealed saturation kinetics and a high temperature dependence. It gave a Km of 85 microM and a Vmax of 962 pmol/min/mg protein and an activation energy (Ea) of 11 kcal/mol. 4. Certain other amino acids (L-valine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan and L-hydroxyproline) significantly stimulated proline uptake. 5. These observations indicate that tsetse fly Glossina morsitans flight muscle mitochondria contain a proline transport mechanism.     

Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

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.

     

Some changes in the flight apparatus of tsetse flies, Glossina morsitans and G. pallidipes, during maturation.

The residual dry weight of the thoracic cuticle of tsetse has been found to increase durint maturation and this finding affects the estimates of mitochondrial and contractile protein made by Bursell. The thoracic proline reserve was also found to increase during maturation, following much the same time course as that of the increases in contractile protein. The results are discussed with reference to the relevant published work on other insects.     

Factors affecting the landing and feeding responses of the tsetse fly Glossina pallidipes to a stationary ox

In Zimbabwe, studies were made of the landing and feeding responses of Glossina pallidipes on an ox. Of the tsetse approaching an ox, ≈ 70% fed. Increasing densities of tsetse increased the grooming responses of the ox but had no significant effect on the percentage of tsetse that engorged. The landing site of tsetse on the ox varied with density, with ≈ 50% landing on the legs at low densities (< 20 flies per ox), compared to ≈ 80% at densities > 40 flies per ox. For male G. pallidipes , the mean bloodmeal size was 37 mg. The probability of feeding was negatively correlated with fat content, declining from 91% for flies with < 1 mg fat to < 50% for flies with > 4 mg fat. Bloodmeal size was also negatively correlated with fat content; the regression equation relating bloodmeal size and fat content indicated that the mean wet weight declined from 42 mg for flies with 1 mg of fat to 31 mg for flies with 5 mg of fat. For females, the probability of feeding was not significantly affected by age as determined by ovarian category but there was a paucity of young (ovarian category 0) flies attracted to the ox. Pregnancy status had no significant effect on the probability of feeding, but samples of flies attracted to the ox showed a relative dearth of females approaching larviposition and a preponderance just after.     

Identification of Tsetse (Glossina spp.) Using Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies'' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the future.     

Tsetse blood-meal sources,endosymbionts and trypanosome-associations in the Maasai Mara National Reserve,a wildlife-human-livestock interface

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.