Identification of Semiochemical Candidates Involved in Glossina Palpalis Gambiensis Larviposition Site Selection and Behavioural Responses of Adult Gravid Females.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of human and animal trypanosomes. This viviparous insect develops and produces a single larva at 10-day intervals deposited in specific sites. In some species aggregation of larvae has been shown and seems to be mediated by both physical factors and volatile semiochemicals of larval origin. In this context, this study aims to identify chemicals emitted during the pupariation process in Glossina palpalis gambiensis. Volatile Organic Compounds (VOCs) emitted by larvae were identified using static headspace solid-phase microextraction and gas-chromatography mass-spectrometry (GC-MS) analysis. Electrophysiology and behavioural assays were performed on gravid females to confirm VOCs behavioural activity and attractiveness. GC-MS results revealed ten chemicals emitted during the pupariation process of G. p. gambiensis larvae. Among these chemicals, gravid females were shown to detect nine of them during coupled gas chromatography - electroantennographic detection tests. Behavioural assays highlighted two compounds were as attractive as pupae and one compound and a blend of four compounds were more attractive than pupae. Although the larval origin of some of them needs to be confirmed as they may also likely produced by micro-organisms, these compounds induced significant behavioural responses in the laboratory. Further experiments have to explore the biological activity and competitiveness of these compounds in the field. This work opens interesting opportunities for behavioural manipulation and control of tsetse flies.

Gamma-radiation of Glossina palpalis gambiensis revisited: effect on fertility and mating competitiveness.

African animal trypanosomoses are vector-borne diseases that cause enormous livestock losses in sub-Saharan Africa, with drastic socio-economic impacts. Vector control in the context of an area-wide integrated pest management program with a sterile insect technique component requires the production of high-quality sterile male tsetse flies. In our study, we evaluated the effect of irradiation on the fecundity of Glossina palpalis gambiensis to identify the optimal dose that will induce maximum sterility while maintaining biological performance as much as possible. In addition, male mating performance was evaluated in semi-field cages. The irradiation doses used were 90, 100, 110, 120, 130, 140, and 150 Gy, and untreated males were used as the control. The results showed that pupal production and emergence rates were higher in batches of females that had mated with fertile males than in those that had mated with irradiated males with any experimental dose. A dose of 120 Gy administered to male flies induced 97-99% sterility after mating with virgin females. For the semi-field cage experiments, males irradiated with 120 Gy showed good sexual competitiveness as compared to fertile males and those irradiated with 140 Gy, considering the level of filling of spermatheca and the number of pairs formed. The optimal radiation dose of 120 Gy found in this study is slightly different from the traditional dose of 110 Gy that has been used in several eradication programmes in the past. The potential reasons for this difference are discussed, and an argument is made for the inclusion of reliable dosimetry systems in these types of studies.

Title: Le rayonnement gamma pour Glossina palpalis gambiensis revisité : effet sur la fertilité et la compétitivité sexuelle. Abstract: Les trypanosomoses animales africaines sont des maladies à transmission vectorielle qui causent d'énormes pertes de bétail en Afrique subsaharienne, avec des impacts socio-économiques importants. La lutte antivectorielle dans le cadre d'un programme de lutte intégrée contre les ravageurs à l'échelle d'une zone avec une composante de technique d'insectes stériles nécessite la production de glossines mâles stériles de haute qualité. Dans notre étude, nous avons évalué l'effet de l'irradiation sur la fécondité de Glossina palpalis gambiensis afin d'identifier la dose optimale qui induira une stérilité maximale tout en maintenant au maximum les performances biologiques. De plus, les performances d'accouplement des mâles ont été évaluées en cages de semi-terrain. Les doses d'irradiation utilisées étaient de 90, 100, 110, 120, 130, 140 et 150 Gy, et des mâles non traités ont été utilisés comme contrôle. Les résultats ont montré que les taux de production et d'émergence de pupes étaient plus élevés dans les lots de femelles qui s'étaient accouplées avec des mâles fertiles que dans les lots de celles accouplées avec des mâles irradiés, avec n'importe quelle dose expérimentale. Une dose de 120 Gy administrée à des mouches mâles a induit une stérilité de 97 à 99 % après accouplement avec des femelles vierges. Pour les expériences en cages de semi-terrain, les mâles irradiés à 120 Gy ont montré une bonne compétitivité sexuelle par rapport aux mâles fertiles et à ceux irradiés à 140 Gy, en considérant le niveau de remplissage de leur spermathèque et le nombre de couples formés. La dose de rayonnement optimale de 120 Gy trouvée dans cette étude est légèrement différente de la dose traditionnelle de 110 Gy qui a été utilisée dans plusieurs programmes d'éradication dans le passé. Les raisons potentielles de cette différence sont discutées et un argument est avancé pour l'inclusion de systèmes de dosimétrie fiables dans ce type d'études.

Quality Control and Mating Performance of Irradiated Glossina palpalis gambiensis Males.

The biological quality of sterile male insects produced in a mass-rearing facility is a prerequisite for the success of the SIT, which is a component of area-wide integrated pest management (AW-IPM). Indeed, sterile male insects released in the field must have a good mating performance in order to compete with wild males, but they must also present the required level of sterility. In the present study, the biological quality of sterile male Glossina palpalis gambiensis produced in a mass-rearing insectary was assessed through quality control testing. The mating performance of irradiated males was assessed in walk-in field cages. Irradiation had no effect on adult emergence but significantly reduced the percentage of operational flies (from 89.58% to 79.87%) and male survival (from 5 to 4 days, on average). However, irradiation did not impact the sterile male insemination potential, with all females inseminated and more than 80% of the spermathecae completely filled. The rate of induced sterility in females was 89.67% due to a dose rate decrease of the radiation source. Moreover, sterile males were able to compete successfully with untreated fertile males for untreated females in walk-in field cages. This study confirmed that the flies were still competitive and stressed the importance of regularly checking the radiation source parameters.

Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso.

BACKGROUND: Animal African Trypanosomosis (AAT) is a parasitic disease of livestock that has a major socio-economic impact in the affected areas. It is caused by several species of uniflagellate extracellular protists of the genus Trypanosoma mainly transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei. In Burkina Faso, AAT hampers the proper economic development of the southwestern part of the country, which is yet the best watered area particularly conducive to agriculture and animal production. It was therefore important to investigate the extent of the infection in order to better control the disease. The objective of the present study was to assess the prevalence of trypanosome infections and collect data on the presence of tsetse flies. METHODS: Buffy coat, Trypanosoma species-specific PCR, Indirect ELISA Trypanosoma sp and trypanolysis techniques were used on 1898 samples collected. An entomological survey was also carried out. RESULTS: The parasitological prevalence of AAT was 1.1%, and all observed parasites were T. vivax. In contrast, the molecular prevalence was 23%, of which T. vivax was predominant (89%) followed by T. congolense (12.3%) and T. brucei s.l. (7.3%) with a sizable proportion as mixed infections (9.1%). T. brucei gambiense, responsible of sleeping sickness in humans, was not detected. The serological prevalence reached 49.7%. Once again T. vivax predominated (77.2%), but followed by T. brucei (14.7%) and T. congolense (8.1%). Seven samples, from six cattle and one pig, were found positive by trypanolysis. The density per trap of Glossina tachinoides and G. palpalis gambiensis was 1.2 flies. CONCLUSIONS/SIGNIFICANCE: Overall, our study showed a high prevalence of trypanosome infection in the area, pointing out an ongoing inadequacy of control measures.

Experimental evidence that immune trypanolysis using the LiTat 1.3 and LiTat 1.5 variant antigen types is not specific to Trypanosoma brucei gambiense in pigs.

In the context of the human African trypanosomiasis elimination process, reliable and accurate diagnostic tools are crucial for exploring the role of a potential animal reservoir of Trypanosoma brucei gambiense. The immune trypanolysis test (TL) using the variant antigen types (VAT) LiTat 1.3 and LiTat 1.5, described as a specific serological method to detect people infected by T. b. gambiense, seems to be a promising tool. However, its specificity was recently questioned during field animal surveys. The present study evaluates the performance of TL during experimental T. b. brucei infection in pigs. Eight infected pigs and four uninfected pigs were followed up with blood and plasma collection. Blood was used for parasitological investigation. TL was performed on the plasma with the LiTat 1.3, LiTat 1.5 and LiTat 1.6 VATs. All control pigs remained negative to parasitological investigation and TL. Trypanosomes were detected in all the infected pigs and the first detection was between 10 and 14 days post infection (dpi). TL results showed that infected pigs developed antibodies against the three VATs. The first antibody detections by TL occurred between 14 and 21 dpi for antibodies directed against LiTat 1.6, 21 and 168 dpi for antibodies directed against LiTat 1.5 and 70, and 182 dpi for antibodies directed against LiTat 1.3. This study highlights for the first time that TL using LiTat 1.3 and LiTat 1.5 VATs is not specific to T. b. gambiense. Development of specific diagnostic tools for the detection of T. b. gambiense infections in animals, especially in pigs, is still needed.

Title: Évidence expérimentale que la trypanolyse basée sur les types d'antigène variable LiTat 1.3 et LiTat 1.5 n'est pas spécifique de Trypanosoma brucei gambiense. Abstract: Dans le contexte d'élimination de la trypanosomiase humaine Africaine, des outils de diagnostic fiables et précis sont essentiels afin d'explorer le rôle d'un potentiel réservoir animal de Trypanosoma brucei gambiense. La trypanolyse (TL) qui utilise les types d'antigène variable (TAV) LiTat 1.3 et LiTat 1.5, et qui est décrite comme une méthode sérologique spécifique pour détecter les personnes infectées par T. b. gambiense, semble être un outil prometteur. Cependant, sa spécificité a été récemment remise en question lors d'enquêtes sur les animaux. La présente étude évalue la performance de ce test lors d'une infection expérimentale à T. b. brucei chez le porc. Huit porcs infectés et quatre porcs témoins non infectés ont été suivis avec des prélèvements de sang et de plasma. Le sang a été utilisé pour l'examen parasitologique. La TL a été réalisée sur les échantillons de plasma avec les TAV LiTat 1.3, LiTat 1.5 et LiTat 1.6. Tous les porcs témoins ont été négatifs en parasitologie et à la TL. Les trypanosomes ont été détectés sur tous les porcs infectés avec les premières détections entre 10 et 14 jours post-infection (jpi). Les résultats de la TL ont montré que les porcs infectés ont développé des anticorps contre les trois TAV. Les premiers anticorps détectés par la TL étaient dirigés contre le LiTat 1.6 entre 14 et 21 jpi, puis le LiTat 1.5 entre 21 et 168 jpi et enfin le LiTat 1.3 entre 70 et 182 jpi. Cette étude démontre pour la première fois que la TL basée sur les TAV LiTat 1.3 et LiTat 1.5 n'est pas spécifique de T. b. gambiense. Il est donc toujours nécessaire et urgent de développer un outil de diagnostic spécifique pour la détection des infections à T. b. gambiense chez les animaux, notamment chez les porcs.

Evaluation of the Re-emergence Risk of Human African Trypanosomiasis in the Southwestern Burkina Faso, A Gold-Bearing Mutation Area.

PURPOSE: The boom in Burkina Faso's artisanal gold mining since 2007 has attracted populations from Côte d'Ivoire and Guinea, which are the West African countries most affected by human African trypanosomiasis (HAT) and therefore increases its risk of re-emergence. Our aim was to update the HAT data in Burkina Faso in the risk of the re-emergence context with the advent of artisanal gold mining. METHODS: The study was carried out in the southwestern Burkina Faso where entomological surveys were conducted using biconical traps in March 2017. Follow by an active medical survey in April 2017, which was targeted the gold panners in 7 villages closer to artisanal gold sites, using CATT, mini-anion exchange centrifugation technique, trypanolysis test (TL) and ELISA test to measure human/tsetse contacts. The buffy coat technique and the TL were also applied in pigs to check their reservoir role of human trypanosomes. RESULTS: Our results have shown no case of HAT among 958 individuals tested and all the 50 pigs were also negative, but the level of antibodies against tsetse saliva evidenced by ELISA revealed low human/tsetse contact. Moreover, gold panners practise agriculture and breeding in an infected tsetse area, which are increased the risk. CONCLUSION: Our results illustrate that the risk of re-emergence is low. The passive surveillance system implemented in 2015 in southwestern Burkina Faso is needed to increase the sentinel sites to better cover this area by taking into account the gold mining. Finally, awareness-raising activities are needed among populations about HAT.

Evaluation of different blood-feeding frequencies on Glossina palpalis gambiensis performance in a mass-rearing insectary.

BACKGROUND: The main challenge to the successful mass-rearing of the tsetse fly in insectaries, especially in Africa, is a sustainable supply of high-quality blood meals. As such, the collection of high-quality blood in large quantities can be an important constraint to production. One possible strategy to lessen the impact of this constraint is to modify the blood-feeding frequency. In the study reported here, we evaluated the effect of three blood-feeding frequencies on the colony performance of Glossina palpalis gambiensis, a riverine tsetse fly species. METHODS: The effect of three, four and six blood-feedings per week on female survival and productivity were evaluated over a 30-day period. Progeny emergence rate and flight ability were also evaluated. RESULTS: Female survival was significantly higher in flies fed four times per week (87%) than in those fed three (72%) and six times per week (78%; P < 0.05). Productivity was similar between flies fed four and six times per week (457 and 454 larvae) but significantly reduced in flies fed three times per week (280 larvae produced; P < 0.05). Both emergence rate and flight ability rate were also similar between flies fed four times per week (97 and 94%, respectively) and six times per week (96 and 97%, respectively), but they were significantly reduced when flies were fed three times per week (89 and 84%, respectively; P < 0.05). CONCLUSIONS: Blood-feeding frequency could be reduced from six times per week to four times per week without affecting mass-rearing production and progeny quality. The implications of these results on tsetse mass-rearing production are discussed.

Neemazal ® as a possible alternative control tool for malaria and African trypanosomiasis?

BACKGROUND: Research efforts to identify possible alternative control tools for malaria and African trypanosomiasis are needed. One promising approach relies on the use of traditional plant remedies with insecticidal activities. METHODS: In this study, we assessed the effect of blood treated with different doses of NeemAzal ® (NA, neem seed extract) on mosquitoes (Anopheles coluzzii) and tsetse flies (Glossina palpalis gambiensis) (i) avidity to feed on the treated blood, (ii) longevity, and (iii) behavioural responses to human and calf odours in dual-choice tests. We also gauged NeemAzal ® toxicity in mice. RESULTS: In An. coluzzii, the ingestion of NA in bloodmeals offered by membrane feeding resulted in (i) primary antifeedancy; (ii) decreased longevity; and (iii) reduced response to host odours. In G. palpalis gambiensis, NA caused (i) a knock-down effect; (ii) decreased or increased longevity depending on the dose; and (iii) reduced response to host stimuli. In both cases, NA did not affect the anthropophilic rate of activated insects. Overall, the most significant effects were observed with NA treated bloodmeals at a dose of 2000 µg/ml for mosquitoes and 50 µg/ml for tsetse flies. Although no mortality in mice was observed after 14 days of follow-up at oral doses of 3.8, 5.6, 8.4 and 12.7 g/kg, behavioural alterations were noticed at doses above 8 g/kg. CONCLUSION: This study revealed promising activity of NA on A. coluzzii and G. palpalis gambiensis but additional research is needed to assess field efficacy of neem products to be possibly integrated in vector control programmes.

Do tsetse flies only feed on blood?

Tsetse flies (Diptera: Glossinidae) are the vectors of trypanosomes causing sleeping sickness in humans, and nagana (animal trypanosomosis) in domestic animals, in Subsaharan Africa. They have been described as being strictly hematophagous, and transmission of trypanosomes occurs when they feed on a human or an animal. There have been indications however in old papers that tsetse may have the ability to digest sugar. Here we show that hungry tsetse (Glossina palpalis gambiensis) in the lab do feed on water and on water with sugar when no blood is available, and we also show that wild tsetse have detectable sugar residues. We showed in laboratory conditions that at a low concentration (0.1%) or provided occasionally (0.1%, 0.5%, 1%), glucose had no significant impact on female longevity and fecundity. However, regular provision of water with 1% glucose increased the mortality and reduced the fecundity of female G. p. gambiensis. The proportion of wild tsetse caught by traps, which have detectable sugar residue in their midgut varied between 5 and 10% according to species (p<10(-3)) and sex, with more females being found with sugar residues than males (p<10(-3)). We also observed a higher frequency of sugar residues in the dry season than in the rainy season (p<10(3)). The infection status did not affect the frequency of sugar residues found (p=0.65), neither did age (p=0.23). These observations represent a fundamental change in our knowledge of this insect vector. They open the way for further research on the field to know more on tsetse feeding behavior regarding other sources of meal than blood, in particular with plants, and may constitute future new means of controlling this vector of neglected tropical diseases.

Detection and identification of pathogenic trypanosome species in tsetse flies along the Comoé River in Côte d'Ivoire.

In order to identify pathogenic trypanosomes responsible for African trypanosomiasis, and to better understand tsetse-trypanosome relationships, surveys were undertaken in three sites located in different eco-climatic areas in Côte d'Ivoire during the dry and rainy seasons. Tsetse flies were caught during five consecutive days using biconical traps, dissected and microscopically examined looking for trypanosome infection. Samples from infected flies were tested by PCR using specific primers for Trypanosoma brucei s.l., T. congolense savannah type, T. congolense forest type and T. vivax. Of 1941 tsetse flies caught including four species, i.e. Glossina palpalis palpalis, G. p. gambiensis, G. tachinoides and G. medicorum, 513 (26%) were dissected and 60 (12%) were found positive by microscopy. Up to 41% of the infections were due to T. congolense savannah type, 30% to T. vivax, 20% to T. congolense forest type and 9% due to T. brucei s.l. All four trypanosome species and subgroups were identified from G. tachinoides and G. p. palpalis, while only two were isolated from G. p. gambiensis (T. brucei s.l., T. congolense savannah type) and G. medicorum (T. congolense forest, savannah types). Mixed infections were found in 25% of cases and all involved T. congolense savannah type with another trypanosome species. The simultaneous occurrence of T. brucei s.l., and tsetse from the palpalis group may suggest that human trypanosomiasis can still be a constraint in these localities, while high rates of T. congolense and T. vivax in the area suggest a potential risk of animal trypanosomiasis in livestock along the Comoé River.

Baited-boats: an innovative way to control riverine tsetse, vectors of sleeping sickness in West Africa.

BACKGROUND: Human African Trypanosomiasis (HAT) is an important neglected tropical disease caused by Trypanosoma spp. parasites transmitted by species of tsetse fly (Glossina spp). The most important vectors of HAT are riverine tsetse and these can be controlled by attracting them to stationary baits such as insecticide-impregnated traps or targets deployed along the banks of rivers. However, the geographical nature of some riverine habitats, particularly mangroves but also extensive lake and river networks, makes deployment of baits difficult and limits their efficacy. It is known that tsetse are attracted by the movement of their hosts. Our hypothesis was that mounting a target on canoes typically used in Africa ('pirogues') would produce an effective means of attracting-and-killing riverine tsetse in extensive wetland habitats. METHODS: In Folonzo, southern Burkina Faso, studies were made of the numbers of tsetse attracted to a target (75 × 50 cm) of blue cloth and netting mounted on a pirogue moving along a river, versus the same target placed on the riverbank. The targets were covered with a sticky film which caught tsetse as they contacted the target. RESULTS: The pirogue-mounted target caught twice as many G. tachinoides and G. p. gambiensis, and 8 times more G. morsitans submorsitans than the stationary one (P < 0.001). CONCLUSION: Pirogues are common vehicle for navigating the rivers, lakes and swamps of West Africa. The demonstration that tsetse can be attracted to targets mounted on such boats suggests that pirogues might provide a cost-effective and convenient platform for deploying targets to control tsetse in the mangrove systems of West Africa where HAT persists. Further studies to assess the impact of pirogue-mounted targets on tsetse populations in HAT foci and the protective value of targets for pirogue passengers are recommended.

A Spatio-temporal Model of African Animal Trypanosomosis Risk.

BACKGROUND: African animal trypanosomosis (AAT) is a major constraint to sustainable development of cattle farming in sub-Saharan Africa. The habitat of the tsetse fly vector is increasingly fragmented owing to demographic pressure and shifts in climate, which leads to heterogeneous risk of cyclical transmission both in space and time. In Burkina Faso and Ghana, the most important vectors are riverine species, namely Glossina palpalis gambiensis and G. tachinoides, which are more resilient to human-induced changes than the savannah and forest species. Although many authors studied the distribution of AAT risk both in space and time, spatio-temporal models allowing predictions of it are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We used datasets generated by various projects, including two baseline surveys conducted in Burkina Faso and Ghana within PATTEC (Pan African Tsetse and Trypanosomosis Eradication Campaign) national initiatives. We computed the entomological inoculation rate (EIR) or tsetse challenge using a range of environmental data. The tsetse apparent density and their infection rate were separately estimated and subsequently combined to derive the EIR using a "one layer-one model" approach. The estimated EIR was then projected into suitable habitat. This risk index was finally validated against data on bovine trypanosomosis. It allowed a good prediction of the parasitological status (r2 = 67%), showed a positive correlation but less predictive power with serological status (r2 = 22%) aggregated at the village level but was not related to the illness status (r2 = 2%). CONCLUSIONS/SIGNIFICANCE: The presented spatio-temporal model provides a fine-scale picture of the dynamics of AAT risk in sub-humid areas of West Africa. The estimated EIR was high in the proximity of rivers during the dry season and more widespread during the rainy season. The present analysis is a first step in a broader framework for an efficient risk management of climate-sensitive vector-borne diseases.

Towards an optimal design of target for tsetse control: comparisons of novel targets for the control of Palpalis group tsetse in West Africa.

BACKGROUND: Tsetse flies of the Palpalis group are the main vectors of sleeping sickness in Africa. Insecticide impregnated targets are one of the most effective tools for control. However, the cost of these devices still represents a constraint to their wider use. The objective was therefore to improve the cost effectiveness of currently used devices. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed on three tsetse species, namely Glossina palpalis gambiensis and G. tachinoides in Burkina Faso and G. p. palpalis in Côte d'Ivoire. The 1 × 1 m(2) black blue black target commonly used in W. Africa was used as the standard, and effects of changes in target size, shape, and the use of netting instead of black cloth were measured. Regarding overall target shape, we observed that horizontal targets (i.e. wider than they were high) killed 1.6-5x more G. p. gambiensis and G. tachinoides than vertical ones (i.e. higher than they were wide) (P < 0.001). For the three tsetse species including G. p. palpalis, catches were highly correlated with the size of the target. However, beyond the size of 0.75 m, there was no increase in catches. Replacing the black cloth of the target by netting was the most cost efficient for all three species. CONCLUSION/SIGNIFICANCE: Reducing the size of the current 1*1 m black-blue-black target to horizontal designs of around 50 cm and replacing black cloth by netting will improve cost effectiveness six-fold for both G. p. gambiensis and G. tachinoides. Studying the visual responses of tsetse to different designs of target has allowed us to design more cost-effective devices for the effective control of sleeping sickness and animal trypanosomiasis in Africa.