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.     

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.     

The salivary secretome of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) infected by salivary gland hypertrophy virus

Background

The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly''s salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.

Methodology/Principal Findings

After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.

Conclusions/Significance

SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.     

Maturation of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) in relation to trap-orientated behaviour

ABSTRACT The fat free dry weight or residual dry weight of the thorax (Trdw) increased linearly for the first 10 days of adult life in both sexes of G.pallidipes in the laboratory as their flight muscles developed. Using ovarian dissection to estimate the ages of nulliparous adult females of G.pallidipes , the Trdw was also found to increase linearly for at least 14 days in the field. Significant increases in pteridine fluorescence with age were measured in both laboratory-reared males and females of known chronological age and in wild-caught nulliparous females whose ages were estimated by ovarian dissection. A linear relationship existed between pteridine fluorescence and wing fray category for a wide range of ages of field-caught flies of both sexes. A stationary trap baited with ox odour was selective in that only the hungrier portion of the flies attracted to it actually entered. However, it was not selective in terms of the mean ages of flies caught. Comparisons were made of the age compositions of catches of both sexes of G.pallidipes attracted to a stationary trap baited with synthetic odours or to a mobile electrified net by plotting Trdw values against pteridine fluorescence. Nulliparous females were not attracted to the stationary trap, but were attracted to the mobile bait. Males of all ages appeared to be equally attracted to both. It is concluded that nulliparous females do not respond to host odour stimuli until they are ready to mate, perhaps relying on the energy-conserving strategy of watching for a moving host animal before attempting to feed. Alternatively, synthetic odours may differ from natural host odours in terms of their attractiveness to young females. Males, however, probably exhibit dual sexual and feeding behaviour by responding to an odour-baited stationary trap even when young.     

Trap efficiency for Glossina pallidipes (Diptera: Glossinidae) at Nguruman, south-west Kenya

An incomplete ring of electric nets was evaluated as a means of estimating trap efficiency for Glossina spp. This methodology assumes flies approach the trap directly, and then enter, or leave directly in random directions. These results showed that the ratio of the number of flies intercepted on the outside of the electric nets to the number on the inside was lower than predicted by this single-approach behavioural model. Moreover, an incomplete ring of nets around a trap reduced trap catch more than the model predicted. These inconsistencies were greater early in the day, and greater for females than for males. It is suggested that flies may make several approaches to a trap before entering or departing. This mixes arriving and departing flies on each side of the electric nets. Use of a complete ring of nets around a trap to estimate trap efficiency entails fewer behavioural assumptions. Catches at a complete ring around a trap were compared to catches in a trap without nets, replicated in a cross-over design. The efficiency of an odour baited NG2G trap was estimated to be 58% for males and 37% for females. Biconical traps were much less efficient. Both trap types were less efficient in the early morning, suggesting entry response is temperature dependent. The NG2G trap was more efficient for non-teneral nulliparous females than for other ages. For both trap types there was little difference between mean fat content of approaching and trapped males, but the mean fat content of trapped females was lower than that of approaching females.     

Thermal tolerance in a south-east African population of the tsetse fly Glossina pallidipes (Diptera, Glossinidae): implications for forecasting climate change impacts

For tsetse (Glossina spp.), the vectors of human and animal trypanosomiases, the physiological mechanisms linking variation in population dynamics with changing weather conditions have not been well established. Here, we investigate high- and low-temperature tolerance in terms of activity limits and survival in a natural population of adult Glossina pallidipes from eastern Zambia. Due to increased interest in chilling flies for handling and aerial dispersal in sterile insect technique control and eradication programmes, we also provide further detailed investigation of low-temperature responses. In wild-caught G. pallidipes, the probability of survival for 50% of the population at low-temperatures was at 3.7, 8.9 and 9.6 degrees C (95% CIs: +/-1.5 degrees C) for 1, 2 and 3 h treatments, respectively. At high temperatures, it was estimated that treatments at 37.9, 36.2 and 35.6 degrees C (95% CIs: +/-0.5 degrees C) would yield 50% population survival for 1, 2 and 3 h, respectively. Significant effects of time and temperature were detected at both temperature extremes (GLZ, p<0.05 in all cases) although a time-temperature interaction was only detected at high temperatures (p<0.0001). We synthesized data from four other Kenyan populations and found that upper critical thermal limits showed little variation among populations and laboratory treatments (range: 43.9-45.0 degrees C; 0.25 degrees C/min heating rate), although reduction to more ecologically relevant heating rates (0.06 degrees C/min) reduce these values significantly from approximately 44.4 to 40.6 degrees C, thereby providing a causal explanation for why tsetse distribution may be high-temperature limited. By contrast, low-temperature limits showed substantial variation among populations and acclimation treatments (range: 4.5-13.8 degrees C; 0.25 degrees C/min), indicating high levels of inter-population variability. Ecologically relevant cooling rates (0.06 degrees C/min) suggest tsetses are likely to experience chill coma temperatures under natural conditions (approximately 20-21 degrees C). The results from acute hardening experiments in the Zambian population demonstrate limited ability to improve low-temperature tolerance over short (hourly) timescales after non-lethal pre-treatments. In flies which survived chilling, recovery times were non-linear with plateaus between 2-6 and 8-12 degrees C. Survival times ranged between 4 and 36 h and did not vary between flies which had undergone chill coma by comparison with flies which had not, even after factoring body condition into the analyses (p>0.5 in all cases). However, flies with low chill coma values had the highest body water and fat content, indicating that when energy reserves are depleted, low-temperature tolerance may be compromised. Overall, these results suggest that physiological mechanisms may provide insight into tsetse population dynamics, hence distribution and abundance, and support a general prediction for reduced geographic distribution under future climate warming scenarios.     

Metabolic rate variation in Glossina pallidipes (Diptera: Glossinidae): gender, ageing and repeatability

Despite the importance of metabolic rate in determining flight time of tsetse and in mediating the influence of abiotic variables on life history parameters (and hence abundance and distribution), metabolic rate measurements and their repeatability have not been widely assessed in these flies. We investigate age-related changes in standard metabolic rate (SMR) and its repeatability, using flow-through respirometry, for a variety of feeding, gender and pregnancy classes during early adult development in laboratory-reared individuals of the tsetse fly, Glossina pallidipes. Standard metabolic rate (144-635 microW) was generally within 22% of previous estimates, though lower than the values found using closed system respirometry. There was no significant difference between the genders, but metabolic rate increased consistently with age, probably owing to flight muscle development. Repeatability of metabolic rate was generally high (r=0.6-.09), but not in younger teneral adults and pregnant females (r approximately equal to 0.05-0.4). In these individuals, low repeatability values are a consequence of muscle or in utero larval development. Tsetse and other flies generally have a much higher metabolic rate, for a given size, than do other insect species investigated to date.     

Artificial larviposition sites for field collections of the puparia of tsetse flies Glossina pallidipes and G. m. morsitans (Diptera: Glossinidae)

Tsetse flies Glossina pallidipes Austen and G. morsitans morsitans Westwood deposit their larvae in warthog burrows, in August-November, at Rekomitjie Research Station, Zambezi Valley, Zimbabwe. Artificial burrows, made from 200-l steel drums, were used to sample these flies and to collect their puparia. Sand-filled plastic trays in the burrows served as a substrate for larval deposition. The sand was covered with c. 2 cm of leaf litter after it was shown that only 3% of larvae were deposited on bare sand if both substrates were available. Other burrow modifications - artificially shading the burrow entrance, increasing the relative humidity inside the burrow, or reducing the size of the burrow entrance - significantly decreased deposition rates. The use of burrows in the hot season results in a reduction in the temperature experienced by the puparium towards an assumed optimum level of 26 degrees C. Artificial burrows maintained a mean temperature of 28.5 degrees C during October-November 1998, c. 2.5 degrees C cooler than ambient; earlier work has shown that natural burrows can be c. 5 degrees C cooler than ambient at these times. This may explain why natural burrows in full sunlight were used for larviposition, whereas artificial burrows were used only when they were in deep shade, and why significantly higher proportions of G. pallidipes were found in natural (66%) than in artificial burrows (34%). Better-insulated artificial burrows might produce more puparia with higher proportions of G. pallidipes. Burrows become waterlogged during the rains and may be too cool for optimum puparial development during the rest of the year. The percentages of G. m. morsitans in catches of females from artificial burrows, refuges and odour-baited traps were 34, 26 and < 10% respectively. Traps are biased in favour of G. pallidipes; artificial burrows may show a bias in favour of G. m. morsitans that is a function of temperature. Artificial warthog burrows provide a convenient way of studying the puparial stage in tsetse and for the first time facilitate the capture of females as they deposit their larvae.     

Assessing mating performance of male Glossina pallidipes (Diptera: Glossinidae) using a walk-in field cage

To monitor the quality of male tsetse for use in the sterile insect technique (SIT), a field cage test was developed and evaluated. Mating competitiveness was tested with male Glossina pallidipes Austen that emerged from pupae stored for different periods at 15 degrees C. Control males emerged from pupae stored at 23-24 degrees C and emerged at 26.5 degrees C. Each sample of test males was divided into two groups with one group being irradiated at 120 Gy; the other group was not irradiated. More than 70% of the maximum possible number of mating pairs occurred in all tests. Males emerged from pupae kept at low temperature and then irradiated formed a greater proportion of mating pairs than the controls. Males emerged from pupae kept at 15 degrees C generally started mating more quickly than the standard colony males although there was no significant difference. Insemination rates were above 99%. Pooled data indicated that mean spermathecal values for females mated with irradiated males were significantly lower than for control males. The duration of copulation varied significantly between treatment groups and was significantly longer for irradiated male flies; there was no correlation between duration of copulation and mean spermathecal value.     

Optimized simulation of the control of tsetse flies Glossina pallidipes and G. m. morsitans (Diptera: Glossinidae) using odour-baited targets in Zimbabwe

In 1984-1985 insecticide-treated targets were deployed in the 600-km2 Rifa Triangle, Zambezi Valley, Zimbabwe. Trap catches of Glossina pallidipes Austen were modelled using a function combining logistic growth with diffusive movement. A simulation routine was linked to a non-linear least-squares optimization programme and fits optimized with respect to population carrying capacities, rates of growth and movement, and to levels of imposed mortality. In March-September 1984, the overall additional mortality was 2% per day of adult female G. pallidipes, increasing thereafter to 8% per day, due to the deployment of more targets, the onset of the hot, dry season and the ground-spraying of the adjoining Zambezi escarpment with DDT. For G. m. morsitans Westwood the corresponding estimates were 1 and 2% per day. For both species, the deployment of four targets km(-2) in a closed population will ensure eradication. For G. m. morsitans a halving of target efficacy would reduce the killing rate to the point where eradication would be unlikely. Estimated daily displacements were c. 200 m for G. m. morsitans and 660 m for G. pallidipes. The lower rate for G. m. morsitans means that, while targets kill this species less effectively, re-invasion of cleared areas is slower. Targets do not markedly affect robust populations outside the deployment area. The Zambian tsetse population adjacent to the Rifa Triangle declined markedly during the experiment, however, suggesting that it is largely maintained by immigration. The methods developed here will be applied to data from other campaigns with the aim of improving the efficiency of tsetse control programmes.     

Mating behaviour of Glossina pallidipes Austen (Diptera, Glossinidae): Genetic differences in copulation time between allopatric populations

Copulation duration of Glossina pallidipes from Lambwe Valley (0° 35, S, 34° 15 E) and Kibwezi Forest (2° 27 S, 37° 55 E) in Kenya, was investigated in the laboratory. The difference in mean copulation time between the populations was highly significant. Reciprocal copulations showed that the trait is predominantly or only determined by the male. F₁ and F₂ male hybrid durations were similar and intermediate between the parental durations. The variances of the F₂ hybrid durations were not significantly larger than those of the F₁ hybrids. Male hybrids from backcrosses had durations on the average intermediate between their parents'. Reciprocal F₁ hybrid male durations gave different means. It is inferred that the trait, in the two populations investigated, is under polygenic control by factors distributed on an unknown number of autosomes and on the X chromosomes.

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Résumé On a étudié et comparé en laboratoire, la durée de copulation de deux populations de Glossina pallidipes du Kenya, l'une de Lambwe Valley (0°35S, 34°15E) l'autre de Kibwezi Forest (2°27S, 37°55E). Les femelles agées de 7–8 jours sont accouplées avec des mâles agés de 14–18 jours, à une température d'environ 27.5° C. La différence de la durée moyenne de copulation entre les deux populations est hautement significative: 21,8 min pour les mouches originaires de Lambwe, contre 59,0 min pour celles originaires de Kibwezi. Des accouplements croisés ont montré que le caractère est avant tout ou uniquement déterminé par le mâle. Les durées de copulation avec des mâles hybrides F₁ et F₂ sont semblables et intermédiaires avec celles observées chez les parents. Les variances de la durée de copulation pour les mâles hybrides F₂ ne sont pas significativement plus grandes que celles des hybrides F₁. Les mâles hybrides issus de croisements de retour offrent des durées de copulation en moyenne intermédiaires avec celles des parents. Les mâles hybrides réciproques F₁ ont des durées de copulation ayant des valeurs moyennes différentes.Il est conclu que le caractère évoqué, dans les deux populations testées, obéit à un contrôle polygénique, dont les facteurs sont distribués à la fois sur un nombre inconnu d'autosomes et sur les chromosomes X. Il est suggéré que le paramètre durée de la copulation peut apporter une information complémentaire dans l'étude systématique des populations de Glossina pallidipes.

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Financed by grants from Uppsala University, The Royal Swedish Academy of Science and The Lars Hierta's Memory Foundation.     

Giemsa C-banding within the genus Glossina (Diptera,Glossinidae)

The Giemsa C-banding staining pattern of members of both the palpalis and morsitans groups of tsetse flies are described. Similarities in the staining reaction of the heterochromatic Y and supernumerary chromosomes within species have been observed which suggests a common origin. The presence of useful markers in all species/sub-species should assist in the recognition of species/sub-species hybrids.     

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%.     

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.     

Does reproductive investment change with age in tsetse flies, Glossina morsitans morsitans (Diptera: Glossinidae)?

1. Viviparous insects such as tsetse ( Glossina spp.) provide unusual opportunities to compare age-related changes in the proportion of maternal resources transferred to offspring.
2. In laboratory populations of Glossina morsitans morsitans the survival of females was high for the first 60 days of adult life but declined rapidly thereafter.
3. Average longevity did not differ significantly between mated and unmated females (93·6 and 90 days, respectively).
4. Nutritional state in terms of fat content and residual dry mass did not decline with adult female age.
5. The fecundity of mated females was constant for the first 60 days of adult life and declined only slightly thereafter.
6. Offspring size did not change towards the end of the adult female lifespan and there was no evidence of an increase in the allocation of resources to reproduction in older females.
7. Results contrast with those obtained recently for vertebrates and may indicate that age-related changes in offspring size in Glossina are not adaptive, or that so few females reach old age under natural conditions that there is no selection for a strategy of terminal investment.     

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.

     

Population genetics of Glossina morsitans submorsitans (Diptera: Glossinidae)

Breeding structure of Glossina morsitans submorsitans Newstead was evaluated by using genetic markers in mitochondrial DNA where diversity was scored at two loci in five natural populations from The Gambia and two populations in Ethiopia (form ugandensis Vanderplank), countries separated by c. 5450 km. Twenty six haplotype combinations were found, of which 17 were shared among two or more populations. Nine haplotypes were found in The Gambia and 23 haplotypes in Ethiopia. There were 12 unique haplotypes. Only six haplotypes were shared between the two countries. Populations in The Gambia (he = 0.26 +/- 0.04) showed less than a third of the diversity of populations in Ethiopia (he = 0.84 +/- 0.03). This suggests recovery from an earlier reduction in population. In a nested analysis of molecular variance of haplotype frequencies, 65% of the variance was due to differences within populations, 34% to differences between populations grouped by country, and only 1% was due to differences among populations within countries. In terms of gene flow, the fixation index FST = 0.35, which leads to an estimate by Wright's island model of less than one reproducing migrant per generation exchanged between the eastern and western submorsitans populations. Nei's genetic similarity measure showed a deep division between Gambian and Ethiopian populations.     

Sexual receptivity and age in Glossina pallidipes Austen (Dipt., Glossinidae)

The recent success of the sterile insect technique (SIT) in eradicating Glossina austeni from Zanzibar has stimulated interest in applying this technology to control Glossina pallidipes. However, little is known about the mating behaviour of this species in relation to the development and implementation of an effective SIT programme. The effect of age on male and female receptivity to mating was evaluated together with copulation duration, sperm transfer and the growth of the accessory gland and follicle A in males and females, respectively. Females and males reached their optimal sexual receptivity 9–13 days after emergence. Mean copulation duration was 20–30 min for mature males and females. The growth of follicle A and the accessory gland (apical body) was a function of age of females and males, respectively. Ovulation was not observed in virgin females up to 15 days of age whereas mated females ovulated by day 9. Males aged 7–15 days were equally effective in inseminating. Cages of males and females of different ages were set up to monitor puparial production in relation to optimization of mass rearing. The results are discussed in relation to the development of an efficient mass rearing protocol for this species and an optimal release strategy for sterile males.     

A white eye color mutant in the tsetse fly Glossina morsitans submotsitans Newstead (Diptera: Glossinidae).

A spontaneous mutation in Glossina morsitans submorsitans Newstead is described. The mutant, designated wht, has white compound eyes but the ocelli and testes have normal coloration. Mutants have lower than normal amounts of xanthommatin and pteridines in their heads. The lesion occurs late in the tryptophan to xanthommatin pathway, in the storage of xanthommatin in the compound eyes, or, most likely, in the transport of precursors into the compound eyes. The locus wht is on the X chromosome.