Prevalence of Trypanosoma and Sodalis in Wild Populations of Tsetse Flies: Impact on Sit Programmes for Tsetse Eradication

Tsetse flies, the vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and Trypanozoon spp were higher in west African countries, whereas tsetse infection with Trypanosoma congolense and T. simiae, T. simiae (tsavo) and T. godfreyi infection prevalence were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in Glossina tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in Glossina palpalis gambiensis, Glossina pallidipes and Glossina medicorum. Trypanosoma infection significantly increased the density of Sodalis in wild G. m. morsitans and G. pallidipes flies however no significant impact of Sodalis infection on trypanosome density.

Interactions Between Glossina Pallidipes Salivary Gland Hypertrophy Virus and Tsetse Endosymbionts in Wild Tsetse Populations

BackgroundTherefore, tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass-rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with micro-organisms, including the primary and obligate Wigglesworthia glossinidia, the commensal Sodalis glossinidius, and Wolbachia pipientis. In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes Salivary Gland Hypertrophy Virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. MethodsIn the present study, we assessed the possible interaction of GpSGHV and tsetse endosymbionts under field conditions to decipher the bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infection with Sodalis and Wigglesworthia density using qPCR. ResultsThe results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans, with an explicit significant negative correlation between GpSGHV and Wigglesworthia infection. GpSGHV infection levels of more than 104 were not observed when Wolbachia infection was present at high density (>108.5), suggesting a potential protective role of Wolbachia against GpSGHV. ConclusionThe result indicates that Wolbachia infection might protect tsetse fly against GpSGHV and the interactions between the tsetse host and its associated microbes are dynamic, likely species-specific and significant differences may exist between laboratory and field conditions.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Satellite-based modelling of potential tsetse (Glossina pallidipes) breeding and foraging sites using teneral and non-teneral fly occurrence data

Background African trypanosomiasis, which is mainly transmitted by tsetse flies (Glossina spp.), is a threat to public health and a significant hindrance to animal production. Tools that can reduce tsetse densities and interrupt disease transmission exist, but their large-scale deployment is limited by high implementation costs. This is in part limited by the absence of knowledge of breeding sites and dispersal data, and tools that can predict these in the absence of ground-truthing. Methods In Kenya, tsetse collections were carried out in 261 randomized points within Shimba Hills National Reserve (SHNR) and villages up to 5 km from the reserve boundary between 2017 and 2019. Considering their limited dispersal rate, we used in situ observations of newly emerged flies that had not had a blood meal (teneral) as a proxy for active breeding locations. We fitted commonly used species distribution models linking teneral and non-teneral tsetse presence with satellite-derived vegetation cover type fractions, greenness, temperature, and soil texture and moisture indices separately for the wet and dry season. Model performance was assessed with area under curve (AUC) statistics, while the maximum sum of sensitivity and specificity was used to classify suitable breeding or foraging sites. Results Glossina pallidipes flies were caught in 47% of the 261 traps, with teneral flies accounting for 37% of these traps. Fitted models were more accurate for the teneral flies (AUC = 0.83) as compared to the non-teneral (AUC = 0.73). The probability of teneral fly occurrence increased with woodland fractions but decreased with cropland fractions. During the wet season, the likelihood of teneral flies occurring decreased as silt content increased. Adult tsetse flies were less likely to be trapped in areas with average land surface temperatures below 24 °C. The models predicted that 63% of the potential tsetse breeding area was within the SHNR, but also indicated potential breeding pockets outside the reserve. Conclusion Modelling tsetse occurrence data disaggregated by life stages with time series of satellite-derived variables enabled the spatial characterization of potential breeding and foraging sites for G. pallidipes. Our models provide insight into tsetse bionomics and aid in characterising tsetse infestations and thus prioritizing control areas. Graphical abstract

Blood meal analysis of tsetse flies (Glossina pallidipes: Glossinidae) reveals higher host fidelity on wild compared with domestic hosts

Background: Changes in climate and land use can alter risk of transmission of parasites between domestic hosts and wildlife, particularly when mediated by vectors that can travel between populations. Here we focused on tsetse flies (genus Glossina), the cyclical vectors for both Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT). The aims of this study were to investigate three issues related to G. palldipes from Kenya: 1) the diversity of vertebrate hosts that flies fed on; 2) whether host feeding patterns varied in relation to type of hosts, tsetse feeding behaviour, site or tsetse age and sex; and 3) if there was a relationship between trypanosome detection and host feeding behaviours or host types. Methods: Sources of blood meals of Glossina pallidipes were identified by sequencing of the mitochondrial cytochrome b gene and analyzed in relationship with previously determined trypanosome detection in the same flies. Results: In an area dominated by wildlife but with seasonal presence of livestock (Nguruman), 98% of tsetse fed on single wild host species, whereas in an area including a mixture of resident domesticated animals, humans and wildlife (Shimba Hills), 52% of flies fed on more than one host species. Multiple Correspondence Analysis revealed strong correlations between feeding pattern, host type and site but these were resolved along a different dimension than trypanosome status, sex and age of the flies. Conclusions: Our results suggest that individual G. pallidipes in interface areas may show higher feeding success on wild hosts when available but often feed on both wild and domesticated hosts. This illustrates the importance of G. pallidipes as a vector connecting the sylvatic and domestic cycles of African trypanosomes.

An update on the distribution of Glossina (tsetse flies) at the wildlife-human-livestock interface of Akagera National Park, Rwanda

Background Glossina (tsetse flies) biologically transmit trypanosomes that infect both humans and animals. Knowledge of their distribution patterns is a key element to better understand the transmission dynamics of trypanosomosis. Tsetse distribution in Rwanda has not been well enough documented, and little is known on their current distribution. This study determined the current spatial distribution, abundance, diversity, and seasonal variations of tsetse flies in and around the Akagera National Park. Methods A longitudinal stratified sampling following the seasons was used. Biconical traps were deployed in 55 sites for 6 consecutive days of each study month from May 2018 to June 2019 and emptied every 48 h. Flies were identified using FAO keys, and the number of flies per trap day (FTD) was used to determine the apparent density. Pearson chi-square (χ2) and parametrical tests (t-test and ANOVA) were used to determine the variations between the variables. The significance (p < 0.05) at 95% confidence interval was considered. Logistic regression was used to determine the association between tsetse occurrence and the associated predictors. Results A total of 39,516 tsetse flies were collected, of which 73.4 and 26.6% were from inside Akagera NP and the interface area, respectively. Female flies accounted for 61.3 while 38.7% were males. Two species were identified, i.e. G. pallidipes [n = 29,121, 7.4 flies/trap/day (FTD)] and G. morsitans centralis (n = 10,395; 2.6 FTD). The statistical difference in numbers was significant between the two species (p = 0.000). The flies were more abundant during the wet season (15.8 FTD) than the dry season (4.2 FTD). Large numbers of flies were trapped around the swamp areas (69.1 FTD) inside the park and in Nyagatare District (11.2 FTD) at the interface. Glossina morsitans was 0.218 times less likely to occur outside the park. The chance of co-existing between the two species reduced outside the protected area (0.021 times). Conclusions The occurrence of Glossina seems to be limited to the protected Akagera NP and a narrow band of its surrounding areas. This finding will be crucial to design appropriate control strategies. Glossina pallidipes was found in higher numbers and therefore is conceivably the most important vector of trypanosomosis. Regional coordinated control and regular monitoring of Glossina distribution are recommended. Graphic Abstract

Characterization of a composite with enhanced attraction to savannah tsetse flies from constituents or analogues of tsetse refractory waterbuck (Kobus defassa) body odor

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck (Kobus defassa) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C5-C10) and 2-alkanones (C8-C13). However, although the blends of carboxylic acids and that of 2-alkanones contributed incrementally to the repellency of the waterbuck odor to savannah tsetse flies, some waterbuck constituents (particularly, nonanoic acid and 2-nonanone) showed significant attractive properties. In another study, increasing the ring size of δ-octalactone from six to seven membered ring changed the activity of the resulting molecule (ε-nonalactone) on the savannah tsetse flies from repellency to attraction. In the present study, we first compared the effect of blending ε-nonalactone, nonanoic acid and 2-nonanone in 1:1 binary and 1:1:1 ternary combination on responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies in a two-choice wind tunnel. The compounds showed clear synergistic effects in the blends, with the ternary blend demonstrating higher attraction than the binary blends and individual compounds. Our follow up laboratory comparisons of tsetse fly responses to ternary combinations with different relative proportions of the three components showed that the blend in 1:3:2 proportion was most attractive relative to fermented cow urine (FCU) to both tsetse species. In our field experiments at Shimba Hills game reserve in Kenya, where G. pallidipes are dominant, the pattern of tsetse catches we obtained with different proportions of the three compounds were similar to those we observed in the laboratory. Interestingly, the three-component blend in 1:3:2 proportion when released at optimized rate of 13.71mg/h was 235% more attractive to G. pallidipes than a combination of POCA (3-n-Propylphenol, 1-Octen-3-ol, 4-Cresol, and Acetone) and fermented cattle urine (FCU). This constitutes a novel finding with potential for downstream deployment in bait technologies for more effective control of G. pallidipes, G. m. morsitans, and perhaps other savannah tsetse fly species, in ‘pull’ and ‘pull-push’ tactics.

Interactions Between Tsetse Endosymbionts and Glossina pallidipes Salivary Gland Hypertrophy Virus in Glossina Hosts

Tsetse flies are the sole cyclic vector for trypanosomosis, the causative agent for human African trypanosomosis or sleeping sickness and African animal trypanosomosis or nagana. Tsetse population control is the most efficient strategy for animal trypanosomosis control. Among all tsetse control methods, the Sterile Insect Technique (SIT) is one of the most powerful control tactics to suppress or eradicate tsetse flies. However, one of the challenges for the implementation of SIT is the mass production of target species. Tsetse flies have a highly regulated and defined microbial fauna composed of three bacterial symbionts (Wigglesworthia, Sodalis and Wolbachia) and a pathogenic Glossina pallidipes Salivary Gland Hypertrophy Virus (GpSGHV) which causes reproduction alterations such as testicular degeneration and ovarian abnormalities with reduced fertility and fecundity. Interactions between symbionts and GpSGHV might affect the performance of the insect host. In the present study, we assessed the possible impact of GpSGHV on the prevalence of tsetse endosymbionts under laboratory conditions to decipher the bidirectional interactions on six Glossina laboratory species. The results indicate that tsetse symbiont densities increased over time in tsetse colonies with no clear impact of the GpSGHV infection on symbionts density. However, a positive correlation between the GpSGHV and Sodalis density was observed in Glossina fuscipes species. In contrast, a negative correlation between the GpSGHV density and symbionts density was observed in the other taxa. It is worth noting that the lowest Wigglesworthia density was observed in G. pallidipes, the species which suffers most from GpSGHV infection. In conclusion, the interactions between GpSGHV infection and tsetse symbiont infections seems complicated and affected by the host and the infection density of the GpSGHV and tsetse symbionts.

An atlas of tsetse and animal African trypanosomiasis in Zimbabwe

Background In the 1980s and 1990s, great strides were taken towards the elimination of tsetse and animal African trypanosomiasis (AAT) in Zimbabwe. However, advances in recent years have been limited. Previously freed areas have been at risk of reinvasion, and the disease in tsetse-infested areas remains a constraint to food security. As part of ongoing control activities, monitoring of tsetse and AAT is performed regularly in the main areas at risk. However, a centralized digital archive is missing. To fill this gap, a spatially explicit, national-level database of tsetse and AAT (i.e. atlas) was established through systematic data collation, harmonization and geo-referencing for the period 2000–2019. Methods The atlas covers an area of approximately 70,000 km2, located mostly in the at-risk areas in the north of the country. In the tsetse component, a total of 33,872 entomological records were assembled for 4894 distinct trap locations. For the AAT component, 82,051 samples (mainly dry blood smears from clinically suspicious animals) were collected at 280 diptanks and examined for trypanosomal infection by microscopy. Results Glossina pallidipes (82.7% of the total catches) and Glossina morsitans morsitans (17.3%) were the two tsetse species recorded in the north and northwest parts of the country. No fly was captured in the northeast. The distribution of AAT follows broadly that of tsetse, although sporadic AAT cases were also reported from the northeast, apparently because of transboundary animal movement. Three trypanosome species were reported, namely Trypanosoma brucei (61.7% of recorded infections), Trypanosoma congolense (28.1%) and Trypanosoma vivax (10.2%). The respective prevalences, as estimated in sentinel herds by random sampling, were 2.22, 0.43 and 0.30%, respectively. Discussion The patterns of tsetse and AAT distributions in Zimbabwe are shaped by a combination of bioclimatic factors, historical events such as the rinderpest epizootic at the turn of the twentieth century and extensive and sustained tsetse control that is aimed at progressively eliminating tsetse and trypanosomiasis from the entire country. The comprehensive dataset assembled in the atlas will improve the spatial targeting of surveillance and control activities. It will also represent a valuable tool for research, by enabling large-scale geo-spatial analyses.