Near-infrared imaging for automated tsetse pupae sex sorting in support of the sterile insect technique.

Tsetse flies are the cyclical vectors of African trypanosomes and one of several methods to manage this vector is the sterile insect technique (SIT). The ability to determine the sex of tsetse pupae with the objective to separate the sexes before adult emergence has been a major goal for decades for tsetse management programmes with an SIT component. Tsetse females develop faster and pharate females inside the pupae melanise 1-2 days before males. This earlier melanisation can be detected by infrared cameras through the pupal shell, and the newly developed Near InfraRed Pupae Sex Sorter (NIRPSS) takes advantage of this. The melanisation process is not homogeneous for all fly organs and the pupa needs to be examined ventrally, dorsally and laterally to ensure accurate classification by an image analysis algorithm. When the pupae are maturing at a constant temperature of 24 °C and sorted at the appropriate age, 24 days post-larviposition for Glossina palpalis gambiensis, the sorting machine can efficiently separate the sexes. The recovered male pupae can then be sterilised for field releases of males, while the rest of the pupae can be used to maintain the laboratory colony. The sorting process with the new NIRPSS had no negative impact on adult emergence and flight ability. A mean male recovery of 62.82 ± 3.61% was enough to provide sterile males to an operational SIT programme, while mean contamination with females (4.69 ± 3.02%) was low enough to have no impact on the maintenance of a laboratory colony.

Title: Imagerie dans l'infrarouge proche pour le tri automatisé du sexe des pupes de glossines comme aide à la technique de l'insecte stérile. Abstract: Les glossines sont les vecteurs cycliques des trypanosomes africains et la technique de l'insecte stérile (TIS) est l'une des méthodes de gestion de ce vecteur. La capacité à déterminer le sexe des pupes de glossines dans le but de séparer les sexes avant l'émergence des adultes a été un objectif majeur, pendant des décennies, pour les programmes de lutte contre les glossines avec une composante TIS. Les femelles tsé-tsé se développent plus rapidement et les pharates femelles à l'intérieur des pupes se mélanisent 1 à 2 jours avant les mâles. Cette mélanisation précoce peut être détectée par des caméras infrarouges à travers la coque de la pupe, ce que le nouveau trieur de sexe des pupes dans le proche infrarouge (TSPPIR) utilise. Le processus de mélanisation n'est pas homogène pour tous les organes de la mouche et la pupe doit être examinée ventralement, dorsalement et latéralement pour assurer une classification précise par un algorithme d'analyse d'image. Lorsque les pupes mûrissent à une température constante de 24 °C et sont triées à l'âge approprié, 24 jours après la larviposition pour Glossina palpalis gambiensis, la machine de tri peut séparer efficacement les sexes. Les pupes mâles récupérées peuvent ensuite être stérilisées pour les lâchers de mâles sur le terrain tandis que le reste des pupes peut être utilisé pour maintenir la colonie de laboratoire. Le processus de tri avec le nouveau TSPPIR n'a eu aucun impact négatif sur l'émergence et la capacité de vol des adultes. Une récupération moyenne des mâles de 62,82 ± 3,61% était suffisante pour fournir des mâles stériles à un programme TIS opérationnel, tandis que la contamination moyenne par les femelles (4,69 ± 3,02%) était suffisamment faible pour n'avoir aucun impact sur le maintien d'une colonie de laboratoire.

Interactions between Glossina pallidipes salivary gland hypertrophy virus and tsetse endosymbionts in wild tsetse populations.

BACKGROUND: 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 microorganisms, 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. METHODS: In the present study, we assessed associations of GpSGHV and tsetse endosymbionts under field conditions to decipher the possible 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 infections with Sodalis and Wigglesworthia density using qPCR. RESULTS: The 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 density. GpSGHV infection levels > 103.31 seem to be absent when Wolbachia infection is present at high density (> 107.36), suggesting a potential protective role of Wolbachia against GpSGHV. CONCLUSION: The result indicates that Wolbachia infection might interact (with an undefined mechanism) antagonistically with SGHV infection protecting tsetse fly against GpSGHV, and the interactions between the tsetse host and its associated microbes are dynamic and likely species specific; significant differences may exist between laboratory and field conditions.

Radiation dose-rate is a neglected critical parameter in dose-response of insects.

Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose-response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose-response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication.

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, 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 analyse 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 T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. 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 G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.

Mitochondrial superoxide dismutase overexpression and low oxygen conditioning hormesis improve the performance of irradiated sterile males.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

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.

A new automated chilled adult release system for the aerial distribution of sterile male tsetse flies.

Tsetse eradication continues to be a top priority for African governments including that of Senegal, which embarked on a project to eliminate Glossina palpalis gambiensis from the Niayes area, following an area-wide integrated pest management approach with an SIT component. A successful SIT programme requires competitive sterile males of high biological quality. This may be hampered by handling processes including irradiation and the release mechanisms, necessitating continued improvement of these processes, to maintain the quality of flies. A new prototype of an automated chilled adult release system (Bruno Spreader Innovation, (BSI™)) for tsetse flies was tested for its accuracy (in counting) and release rate consistency. Also, its impact on the quality of the released sterile males was evaluated on performance indicators, including flight propensity, mating competitiveness, premating and mating duration, insemination rate of mated females and survival of male flies. The BSITM release system accurately counted and homogenously released flies at the lowest motor speed set (0.6 rpm), at a consistent rate of 60±9.58 males/min. Also, the release process, chilling (6 ± 1°C) and passing of flies through the machine) had no significant negative impact on the male flight propensity, mating competitiveness, premating and mating durations and the insemination rates. Only the survival of flies was negatively affected whether under feeding or starvation. The positive results of this study show that the BSI™ release system is promising for use in future tsetse SIT programmes. However, the negative impact of the release process on survival of flies needs to be addressed in future studies and results of this study confirmed under operational field conditions in West Africa.

Modified Atmosphere Does Not Reduce the Efficacy of Phytosanitary Irradiation Doses Recommended for Tephritid Fruit Flies.

Phytosanitary irradiation (PI) has been successfully used to disinfest fresh commodities and facilitate international agricultural trade. Critical aspects that may reduce PI efficacy must be considered to ensure the consistency and effectiveness of approved treatment schedules. One factor that can potentially reduce PI efficacy is irradiation under low oxygen conditions. This factor is particularly important because storage and packaging of horticultural commodities under low oxygen levels constitute practices widely used to preserve their quality and extend their shelf life. Hence, international organizations and regulatory agencies have considered the uncertainties regarding the efficacy of PI doses for insects infesting fresh commodities stored under low oxygen levels as a rationale for restricting PI application under modified atmosphere. Our research examines the extent to which low oxygen treatments can reduce the efficacy of phytosanitary irradiation for tephritids naturally infesting fruits. The effects of normoxia (21% O2), hypoxia (~5% O2), and severe hypoxia (< 0.5% O2) on radiation sensitivity of third instars of Anastrepha fraterculus (sensu lato), A. ludens (Loew), Bactrocera dorsalis (Hendel), and Ceratitis capitata (Wiedemann) were evaluated and compared at several gamma radiation doses. Our findings suggest that, compared to normoxia, hypoxic and severe-hypoxic conditioning before and during irradiation can increase adult emergence and contribute to advancement of larval development of tephritid fruit flies only at low radiation doses that are not used as phytosanitary treatments. With phytosanitary irradiation doses approved internationally for several tephritids, low oxygen treatments applied before and during irradiation did not increase the emergence rates of any fruit fly species evaluated, and all treated insects died as coarctate larvae. Thus, the findings of our research support a re-evaluation of restrictions related to phytosanitary irradiation application under modified atmospheres targeting tephritid fruit flies.

The role of oxygen depletion and subsequent radioprotective effects during irradiation of mosquito pupae in water.

BACKGROUND: Radiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in water versus in air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions. METHODS: Pupae of Aedes aegypti, Ae. albopictus, and Anopheles arabiensis were submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae of Ae. aegypti (aged 40-44 h), Ae. albopictus (aged 40-44 h) and An. arabiensis (aged 20-24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with < 0.5% O2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility. RESULTS: All three species depleted the water of DO to levels under 0.5% within 30 minutes, with An. arabiensis consuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses (P < 0.0001), increasing at higher doses. This effect was most pronounced in An. arabiensis. CONCLUSIONS: The consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Tolerance to a Combination of Low Temperature and Sterilizing Irradiation in Male Glossina palpalis gambiensis (Diptera: Glossinidae): Simulated Transport and Release Conditions.

Recently, aerial delivery of sterilized adult tsetse flies has been developed based on the release of chilled adult sterile males. The long-distance transport of irradiated male tsetse pupae for chilled adult release systems requires exposure of the mature pupae to irradiation and to low temperatures for both the pupae and adults. The effect of these treatments on mating of adult Glossina palpalis gambiensis (Vanderplank, Diptera: Glossinidae) males was investigated. Male G. p. gambiensis pupae were stored at 10°C for 5 d and irradiated with 110 Gy within the first 24 h of cold storage. In addition, to simulate a chilled adult release environment, 6-d-old adult males were stored at 5.1 ± 0.4°C for 6 or 30 h. Mating performance was compared to untreated controls in walk-in field cages. A significantly lower proportion of males that had been irradiated and stored at low temperature succeeded in securing a mating compared to untreated males. Premating time, copulation duration and spermathecal fill were similar. Insemination levels were slightly lower for adult males stored at low temperature for 30 h compared to 6 h or control. Although the mating behavior of the males was affected by the treatments given, the data presented confirm the suitability of using long-distance transport of chilled and irradiated male G. p. gambiensis pupae followed by releasing the emerged adult male flies using a chilled adult release system. However, the data indicate that the chilling duration of the adults should be minimized.

Incompatible and sterile insect techniques combined eliminate mosquitoes.

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

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.

Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of Sodalis glossinidius in tsetse flies.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. RESULTS: Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. CONCLUSION: Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach - using modified Sodalis to produce males refractory to trypanosome infection - with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes.

High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.).

BACKGROUND: In African tsetse flies Glossina, spp. detection of bacterial symbionts such as Wolbachia is challenging since their prevalence and distribution are patchy, and natural symbiont titers can range at levels far below detection limit of standard molecular techniques. Reliable estimation of symbiont infection frequency, especially with regard to interrelations between symbionts and their potential impact on host biology, is of pivotal interest in the context of future applications for the control and eradication of Glossina-vectored African trypanosomosis. The presence or absence of symbionts is routinely screened with endpoint polymerase chain reaction (PCR), which has numerous advantages, but reaches its limits, when detecting infections at natural low titer. To not only determine presence of native tsetse symbionts but also to localize them to specific host tissues, fluorescence in situ hybridization (FISH) can be applied. However, classic FISH assays may not detect low-titer infections due to limitations in sensitivity. RESULTS: We have compared classic endpoint PCR with high-sensitivity blot-PCR. We demonstrate that the latter technique allows for clear detection of low-titer Wolbachia in the morsitans and palpalis groups while classic endpoint PCR does not. In order to localize Wolbachia in situ in high and low-titer Glossina species, we applied high-end Stellaris® rRNA-FISH. We show that with this high sensitivity method, even low amounts of Wolbachia can be traced in specific tissues. Furthermore, we highlight that more tissues and organs than previously recorded are infested with Wolbachia in subspecies of the morsitans and palpalis groups. CONCLUSIONS: Our results demonstrate that overall symbiont infection frequencies as well as the presence in specific host tissues may be underestimated when using low-sensitivity methods. To better understand the complex interrelation of tsetse flies and their native symbionts plus the pathogenic trypanosomes, it is important to consider application of a broader range of high-sensitivity detection tools.

Prevalence of trypanosomes, salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal. RESULTS: The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected. CONCLUSION: The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control. RESULTS: In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species. CONCLUSIONS: The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

Hytrosavirus genetic diversity and eco-regional spread in Glossina species.

BACKGROUND: The management of the tsetse species Glossina pallidipes (Diptera; Glossinidae) in Africa by the sterile insect technique (SIT) has been hindered by infections of G. pallidipes production colonies with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae family). This virus can significantly decrease productivity of the G. pallidipes colonies. Here, we used three highly diverged genes and two variable number tandem repeat regions (VNTRs) of the GpSGHV genome to identify the viral haplotypes in seven Glossina species obtained from 29 African locations and determine their phylogenetic relatedness. RESULTS: GpSGHV was detected in all analysed Glossina species using PCR. The highest GpSGHV prevalence was found in G. pallidipes colonized at FAO/IAEA Insect Pest Control Laboratory (IPCL) that originated from Uganda (100%) and Tanzania (88%), and a lower prevalence in G. morsitans morsitans from Tanzania (58%) and Zimbabwe (20%). Whereas GpSGHV was detected in 25-40% of G. fuscipes fuscipes in eastern Uganda, the virus was not detected in specimens of neighboring western Kenya. Most of the identified 15 haplotypes were restricted to specific Glossina species in distinct locations. Seven haplotypes were found exclusively in G. pallidipes. The reference haplotype H1 (GpSGHV-Uga; Ugandan strain) was the most widely distributed, but was not found in G. swynnertoni GpSGHV. The 15 haplotypes clustered into three distinct phylogenetic clades, the largest contained seven haplotypes, which were detected in six Glossina species. The G. pallidipes-infecting haplotypes H10, H11 and H12 (from Kenya) clustered with H7 (from Ethiopia), which presumably corresponds to the recently sequenced GpSGHV-Eth (Ethiopian) strain. These four haplotypes diverged the most from the reference H1 (GpSGHV-Uga). Haplotypes H1, H5 and H14 formed three main genealogy hubs, potentially representing the ancestors of the 15 haplotypes. CONCLUSION: These data identify G. pallidipes as a significant driver for the generation and diversity of GpSGHV variants. This information may provide control guidance when new tsetse colonies are established and hence, for improved management of the virus in tsetse rearing facilities that maintain multiple Glossina species.

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes.

BACKGROUND: Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host's mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene). RESULTS: We show that in response to hemocoelic injections of GpSGHV into G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes. CONCLUSION: The upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.