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.     

Factors affecting density-independent survival of an island population of tsetse flies in Zimbabwe

Analysis is presented of the factors affecting survival probability in populations of tsetse flies Glossina morsitans morsitans Westwood and G. pallidipes Austen (Diptera: Glossinidae) on Antelope Island, Lake Kariba, Zimbabwe. For mature male and female adult G. m. morsitans mean temperature (T _bar) accounted for 70 and 50%, respectively, of the variance in mark-recapture estimates of survival when the flies were not subjected to trapping. Saturation deficit (SD) only accounted for 36 and 33%, respectively. Maximum temperature (T_max) and SD accounted for 36–42% of the variance in male and female G. pallidipes. For the corresponding Moran curve estimates of the survival over all developmental stages, SD lagged by three weeks accounted for 61 and 41% of the variance for male and female G. m. morsitans, respectively, and 64 and 56% for G. pallidipes. The corresponding figures for plots against T_max were 44, 23, 23, and 21%, respectively. The same patterns were seen in the whole data set once allowance was made for the effect of trapping on survival and for an effect of season, correlated with an index of photosynthetic activity. For male G. m. morsitans there was a significant effect of saturation deficit, but not temperature, on immature survival. Decreased adult survival at high temperatures results from the need to feed more frequently and hence to take more risks per unit time. High saturation deficits result directly in reduced emergence of healthy flies from pupae.     

Insect immunity: Induction of cecropin and attacin-like antibacterial factors in the haemolymph of Glossina morsitans morsitans

Injections of live Escherichia coli into adult tsetse flies, Glossina morsitans morsitans induced an antibacterial activity in the haemolymph after a lag period of 6–18 hr. Peak activity occurred after 24–72 hr with a dose of 10⁴ bacteria/fly. Acidic electrophoresis of immune haemolymph from G. m. morsitans followed by an antibacterial assay on the gel revealed the presence of cecropin- and attacin-like factors. The induction of antibacterial activity in tsetse was completely blocked by injection of cycloheximide, a known inhibitor of protein synthesis in eukaryotic organisms. Purified InA from Bacillus thuringiensis, a proteolytic enzyme with specificity for cecropins and attacins in haemolymph, inactivated the antibacterial activity in tesetse immune haemolymph. When tested against 10 different bacterial species, the spectrum was the same for the antibacterial activity in immune haemolymph from tsetse and Cecropia.     

Seasonal variation of tsetse fly species abundance and prevalence of trypanosomes in the Maasai Steppe,Tanzania

Tsetse flies, the vectors of trypanosomiasis, represent a threat to public health and economy in sub‐Saharan Africa. Despite these concerns, information on temporal and spatial dynamics of tsetse and trypanosomes remain limited and may be a reason that control strategies are less effective. The current study assessed the temporal variation of the relative abundance of tsetse fly species and trypanosome prevalence in relation to climate in the Maasai Steppe of Tanzania in 2014–2015. Tsetse flies were captured using odor‐baited Epsilon traps deployed in ten sites selected through random subsampling of the major vegetation types in the area. Fly species were identified morphologically and trypanosome species classified using PCR. The climate dataset was acquired from the African Flood and Drought Monitor repository. Three species of tsetse flies were identified: G. swynnertoni (70.8%), G. m. morsitans (23.4%), and G.pallidipes (5.8%). All species showed monthly changes in abundance with most of the flies collected in July. The relative abundance of G. m. morsitans and G. swynnertoni was negatively correlated with maximum and minimum temperature, respectively. Three trypanosome species were recorded: T. vivax (82.1%), T. brucei (8.93%), and T. congolense (3.57%). The peak of trypanosome infections in the flies was found in October and was three months after the tsetse abundance peak; prevalence was negatively correlated with tsetse abundance. A strong positive relationship was found between trypanosome prevalence and temperature. In conclusion, we find that trypanosome prevalence is dependent on fly availability, and temperature drives both tsetse fly relative abundance and trypanosome prevalence.     

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix

Background

Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.

Methods

PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.

Results

Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.

Conclusion

To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.     

Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina species offers an opportunity to compare their chemosensory repertoire and enhance our understanding of their biology in relation to chemosensation. Here, we identified and annotated the major chemosensory gene families in Glossina. We identified a total of 118, 115, 124, and 123 chemosensory genes in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. pallidipes, respectively, relative to 127 reported in G. m. morsitans. Our results show that tsetse fly genomes have fewer chemosensory genes when compared to other dipterans such as Musca domestica (n>393), Drosophila melanogaster (n = 246) and Anopheles gambiae (n>247). We also found that Glossina chemosensory genes are dispersed across distantly located scaffolds in their respective genomes, in contrast to other insects like D. melanogaster whose genes occur in clusters. Further, Glossina appears to be devoid of sugar receptors and to have expanded CO₂ associated receptors, potentially reflecting Glossina''s obligate hematophagy and the need to detect hosts that may be out of sight. We also identified, in all species, homologs of Ir84a; a Drosophila-specific ionotropic receptor that promotes male courtship suggesting that this is a conserved trait in tsetse flies. Notably, our selection analysis revealed that a total of four gene loci (Gr21a, GluRIIA, Gr28b, and Obp83a) were under positive selection, which confers fitness advantage to species. These findings provide a platform for studies to further define the language of communication of tsetse with their environment, and influence development of novel approaches for control.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Phylogenetically distant symbiotic microorganisms reside in Glossina midgut and ovary tissues

Abstract. Many blood-feeding insects, including tsetse flies (Diptera: Glossinidae), harbour intracellular bacterial symbionts. Using isolates from tissues of several Glossina species and diagnostic DNA oligonucleotide primers, a polymerase chain reaction (PCR) based assay was designed to identify symbiotic bacteria. Those inhabiting the midgut of Glossina were found to belong to the gamma subdivision, whereas ovarian Proteobacteria were of the alpha subdivision - probably genus Wolbachia (Rickettsiaceae). The presence of Wolbachia-like Rickettsia in the ovaries of G. morsitans subspecies may help to explain the maternally inherited incompatibility of some crosses within this species.     

“Wigglesworthia morsitans” Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between “Wigglesworthia morsitans” (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B₉) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control.     

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.     

Precocene-induced sterility in F1 generation of Glossina morsitans morsitans

Precocene treatment does not disrupt the events of reproduction in Glossina morsitans morsitans or induce any apparent changes in treated tsetse. However, some females of the F₁ generation are either sterile or show retardations in follicle development. Sterility is not reversed spontaneously or with juvenile hormone analogues. The critical period for precocene action is related to each ovulation. The corpora allata of precocene-treated tsetse are normal, but those of F₁ sterile females are degenerate. The occurrence of retardation has enabled the characterisation of stages in follicle development in G. m. morsitans.     

A Neglected Aspect of the Epidemiology of Sleeping Sickness: The Propensity of the Tsetse Fly Vector to Enter Houses

Background

When taking a bloodmeal from humans, tsetse flies can transmit the trypanosomes responsible for sleeping sickness, or human African trypanosomiasis. While it is commonly assumed that humans must enter the normal woodland habitat of the tsetse in order to have much chance of contacting the flies, recent studies suggested that important contact can occur due to tsetse entering buildings. Hence, we need to know more about tsetse in buildings, and to understand why, when and how they enter such places.

Methodology/Principal Findings

Buildings studied were single storied and comprised a large house with a thatched roof and smaller houses with roofs of metal or asbestos. Each building was unoccupied except for the few minutes of its inspection every two hours, so focusing on the responses of tsetse to the house itself, rather than to humans inside. The composition, and physiological condition of catches of tsetse flies, Glossina morsitans morsitans and G. pallidipes, in the houses and the diurnal and seasonal pattern of catches, were intermediate between these aspects of the catches from artificial refuges and a host-like trap. Several times more tsetse were caught in the large house, as against the smaller structures. Doors and windows seemed about equally effective as entry points. Many of the tsetse in houses were old enough to be potential vectors of sleeping sickness, and some of the flies alighted on the humans that inspected the houses.

Conclusion/Significance

Houses are attractive in themselves. Some of the tsetse attracted seem to be in a host-seeking phase of behavior and others appear to be looking for shelter from high temperatures outside. The risk of contracting sleeping sickness in houses varies according to house design.     

Age‐specific changes in sperm levels among female tsetse (Glossina spp.) with a model for the time course of insemination

The age, insemination and ovulation status of tsetse flies Glossina pallidipes Austen (n = 154369) and Glossina morsitans morsitans Westwood (n = 19659), captured over 11 years in Zimbabwe, are assessed by ovarian dissection. Instantaneous rates of insemination increase exponentially with age in both species; 90% insemination levels are reached after 5 days post‐emergence in G. m. morsitans and 7 days in G. pallidipes, varying little with season. More than 95% of both species have ovulated by the age of 8 days and 99% by 12 days. Older flies that have not ovulated are > 100‐fold more likely to be caught in October and November than in other months. A 500‐fold decrease in trap catches did not result in any detectible decrease in the probability of females being inseminated. The proportion of partially filled spermathecae rises for approximately 6 days then declines, consistent with some flies having mated more than once. For flies caught on electric nets, with wings undamaged during capture, wing‐fray data are used to extend ovarian age estimates up to 11 ovulations. Among these flies, the volume of sperm in the spermathecae declines little in flies that have ovulated up to seven times; thereafter, it declines by approximately 1% per ovulation. The time course of insemination and the mating frequency of females are important considerations in modelling tsetse fly populations, as well as for the dynamics of interventions involving the release of genetically‐modified insects, which should not be seriously compromised by the limited levels of polyandry currently observed.     

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (<Emphasis Type="Italic">Glossina m. morsitans</Emphasis>)

Background

Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host’s nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography – mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies.

Results

All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays.

Conclusions

While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

     

The diurnal activity, movement and trypanosome infection rates of Glossina fuscipes fuscipes （Diptera： Glossinidae） in Buvuma Island, Lake Victoria, Uganda

The diurnal activity patterns, trypanosome infection rates and movement of Glossinafuscipesfuscipes （Diptera： Glossinidae） were investigated in Buvuma Island, Lake Victoria, Uganda. Hourly trapping of tsetse flies was undertaken to determine their activity rhythm while a capture-mark-release-recapture method was conducted to assess the movement and dispersal of tsetse flies between lakeshore, hinterland and further inland sites along a transected area. Dissection of tsetse flies was also undertaken to determine the trypanosome infection rates in salivary glands, proboscis and mid-gut. Results indicated a bimodal diurnal activity profile for G. f fuscipes on the Island, both on the lakeshore and in the hinterland. Movement and dispersal of G. f fuscipes tsetse flies occurred between lakeshore, hinterland and further inland sites with a greater tendency of flies to move to the lakeshore. Trypanosome infection rates of 4.32% for Trypasoma vivax and 1.15% for 7. congolense were found in G. f. fuscipes.     

The use of discriminant analysis for the estimation of sampling biases for female tsetse

ABSTRACT.

• 1 In this paper we investigate whether the technique of discriminant analysis can be used to estimate sampling biases for female tsetse.
• 2 Discriminant analysis was first applied to laboratory samples of female tsetse, Glossina morshans morsitans Westwood, to test whether flies of known history could be assigned to the correct day of the pregnancy cycle on the basis of their fat, haematin and corrected residual dry weight.
• 3 Following the satisfactory results from the laboratory samples, the same technique was applied to field samples of G.m. centralis Machado captured by electric traps and hand nets in Zambia and of G.palpalis palpalis (Robineau-Desvoidy) captured in biconical traps at five sites in Ivory Coast. The results show that flies on day 1 of the pregnancy cycle were most likely to be caught, with a second peak of day-6 and day-7 flies, while very few day-8 or day-9 flies were caught.
• 4 These major peaks in fly trappability coincide with the known feeding habits of female tsetse, and indicate synchrony of feeding by many members of the population immediately after larviposition and again as the larva in utero moults from the second to third instar. G.palpalis is relatively more available at this later stage of its pregnancy cycle to the capture methods used than is G.morsitans. A third feed may be taken at a more variable point in the pregnancy cycle.
• 5 This method of estimating the sampling biases of female tsetse could allow an estimate of total population size, as long as the absolute sampling efficiency of flies on any one day of the pregnancy cycle could be established by, for example, mark-release-recapture experiments.

     

Glycogen in the proventriculus of the tsetse fly

Glycogen was detected in the proventriculus of the tsetse fly, Glossina morsitans morsitans, by ultrastructural, histochemical, and biochemical methods. This organ contained ten times or more glycogen on a dry weight basis than was found in the thoracic muscle. Proventriculi of male tsetse contained less glycogen than those of females belonging to the same age group and in teneral flies the amount of glycogen was about 50 per cent lower than in mature, fed flies of the same sex. Although the thoracic muscle of tsetse flies was considerably lower in glycogen than that of blowflies the amounts in the proventriculus of mature females of the two insect species were almost equal. It is suggested that this carbohydrate store may supply the energy required for secretory processes.