Tsetse flies (Diptera: Glossinidae) are the main vectors of animal and human trypanosomoses in Africa. The Sterile Insect Technique (SIT) has proven effective in controlling tsetse flies

when applied to isolated populations but necessitates the production of large numbers of sterile males. A new approach, called boosted SIT, combining SIT with the contamination of wild

females by sterile males coated with biocides has been proposed for large-scale control of vector populations. The aim of the study was to evaluate this new approach using pyriproxyfen on

the riverine species Glossina palpalis gambiensis (Vanderplank, 1949) in the laboratory. The contamination dose and persistence of pyriproxyfen on sterile males, the impact of pyriproxyfen

pyriproxyfen has no impact on the survival of sterile males. Pyriproxyfen persisted on sterile males for up to 10 days at a dose of 100 ng per fly. In addition, the horizontal transfer of

pyriproxyfen from a treated sterile male to a female during mating could be measured with an average of 50 ng of pyriproxyfen transferred. After contacts without mating, the average quantity

transferred was more than 10 ng. Finally, the pyriproxyfen powder was very effective on G. p. gambiensis leading to 0% emergence of the pupae produced by contaminated females. These

promising results must be confirmed in the field. A large-scale assessment of this boosted pyriproxyfen-based SIT approach will be carried out against tsetse flies in Senegal (West Africa).

On the African continent, tsetse flies (Diptera: Glossinidae) are the main vectors of the parasites responsible for Human African Trypanosomosis (HAT), or sleeping sickness, and Animal

African Trypanosomosis (AAT), also called Nagana1. These diseases caused by Trypanosomatidae belong to the group of neglected tropical diseases (NTDs), occurring in developing areas2.

Sub-Saharan African countries suffer from a significant impact of NTDs on public health and economic development3. Glossina palpalis gambiensis (Vanderplank, 1949) is one of the main vectors

of the HAT parasite in West Africa and is responsible for the persistence of many residual outbreaks of this disease in forest and savannah areas4. There is still no vaccine against HAT and

curative treatments are difficult to access for the most exposed and vulnerable populations5. Glossina palpalis gambiensis is also involved in the transmission of AAT, which affects

livestock and reduces animal production thus limiting the availability of food resources6,7. Despite the millions of doses of trypanocides administered, nearly 3 million cattle die each year

in Africa from AAT. Annual direct and indirect agricultural losses attributed to this disease are estimated at more than US$ 4 billion8. In addition, many wild bovids infected with

trypanosomes do not suffer from serious clinical disease and are thus an important reservoir of infection. The presence of tsetse flies in Africa limits access to millions of square

kilometres of fertile and resource-rich land7.

To limit the impact of trypanosomoses in Africa, it is necessary to interrupt the trypanosome transmission cycle by reducing the host-vector contact by controlling tsetse fly populations.

This control can be achieved through a variety of techniques9, including traps, insecticide impregnated screens10, live bait technique11,12, sequential aerosol technique13, and the sterile

insect technique14 (SIT).

SIT is based on the mass release of irradiated sterile males. This genetic control technique has shown interesting results in controlling tsetse flies15. Irradiation causes a multitude of

random dominant lethal mutations, leading to infertility16. Sterilized males compete for mating with fertile wild females, reducing population fecundity and causing population collapse17.

SIT proved effective in 1997 on the Island of Unguja (Tanzania) where it eradicated the local vector of AAT, Glossina austeni (Newstead, 1912), allowing livestock development14,15,16,17,18.

SIT is most effective when the wild population is isolated and its density has already been reduced by trapping or using insecticide-impregnated screens or sequential aerial technique15.

Nevertheless, the release of sterile insects is effective provided that sterile males have the ability to fly, survive and compete with their wild counterparts and that sufficient ratio of

sterile to wild males can be achieved19,20. The successful mating between sterile males and wild fertile females is mandatory for a successful SIT program. It is estimated that an average

ratio of 10 sterile males released to one wild male would compensate for the lower competitiveness of sterile males compared to wild males in the field21.

However, SIT faces many technical and logistical constraints, particularly when it comes to treating tsetse populations over large areas such as in continental Africa21. This is why a new

control method called “boosted SIT” has been proposed. It combines SIT and the self-dissemination of a biocide by the insect22. The principle of self-dissemination of a biocide is to allow

the horizontal transfer of this biocide between two insects of the same species, for example from the male to the female through their mating23. The hypothesis is that a simple contact

between sterile males and wild females is enough to disseminate the biocide even if the mating attempts fails.

Here, the biocide is a juvenile hormone analogue, pyriproxyfen (PP), which acts by mimicking the action of the juvenile insect hormone24. Like other insect growth regulators (IGRs), PP is

toxic to a broad spectrum of insects during their developmental stages. Pyriproxyfen has traditionally been used in aquatic habitats to prevent mosquito larvae and pupae from developing into

adults25. In addition, PP also has an impact on fecundity and fertility of adult mosquito females through tarsal contact. Pyriproxyfen affects both the production and development of eggs

and adult emergence26,27. Studies on Aedes mosquitoes have showed that exposure to PP can reduce the reproductive capacity of mosquito females28,29,30. Moreover, a study conducted with A.

aegypti has shown that PP could be transferred from treated males to virgin females during mating, and then subsequently transferred from females to water-holding containers at

concentrations that inhibited emergence31. For tsetse flies, adult females of Glossina morsitans morsitans (Wiedemann, 1850), exposed to a dose as low as 0.01 µg of PP produced non-viable

offspring for at least two reproductive cycles32. The same study showed that males of G. m. morsitans exposed to 0.1 µg of PP could transfer a sufficient dose during mating females to cause

sterility in their mates.

Pyriproxyfen has no mutagenic or toxic effect on mammals at the doses recommended by WHO since it targets an insect-specific metabolic pathway33. It is used against pests of public health

interest (houseflies, cockroaches), agricultural pests (aphids, whiteflies) and pathogen-carrying insects24 (mosquitoes) for which it is authorized in drinking water33. In addition, no

resistance of tsetse flies to this biocide is currently known. The Boosted SIT method applied to tsetse consists of the mass release of male tsetse flies sterilized by radiation and coated

with biopesticides, in this case PP, into wild tsetse fly populations. Thus, males will sterilize not only wild virgin females, but also already inseminated females. Previously inseminated

females generally refuse to mate again, but may still receive a biocide during mating attempts of treated sterile males. Indeed, sexual harassment of females, even those already inseminated,

by males is common among tsetse and even causes increased mortality34.

The technique of contaminating sterile males with PP is an important part of this new approach. We assume that PP is more easily transferred to the female during mating when it is located on

the outer part of the male’s body. Once on the female, part of the PP can be transferred to the larva in utero affecting its development as shown previously32. In addition, it is possible

that a quantity of PP, present on the female, may be deposited on the larva during delivery. Thus, the biocide could have an impact on pupal development and adult emergence35.

This innovative method could be used to treat sterile tsetse males before their aerial release by aircraft or by drones36. The ultimate goal of the project is to eliminate targeted

populations of riverine tsetse flies in at risk areas of tropical Africa, after conducting epidemiological and socio-economic studies on these populations.

The goal of our study was to evaluate in G. p. gambiensis, the impact of a solid PP formulation on sterile male survival, its persistence on treated males and its transfer rate to females

during mating as well as its impact on their progeny production.

Survival rate of PP-treated sterile males remained stable for the first three days of the experiment, then decreased until 21 days’ post treatment, staying above 75% (Fig. 1). The control

flies’ survival rate (black line) was similar to the average survival rate of PP-treated groups (blue line) for the first 10 days, then fell below 65% until day 21 of the experiment. No

significant difference in survival rate was noted between treated and control groups (Cox test: p = 0.55). Unexpectedly, the survival rate of the control treated with the F15 carrier powder

only (formulation without PP) (black dash line) dropped rapidly to less than 25% only 24 hours after treatment.

Survival rate of sterile males of Glossina palpalis gambiensis over 21 days. The control group is composed of n = 50 untreated flies and the control [F15] group of n = 50 flies treated with

powder F15. The test [F15 + PP] represents the average survival rate of five groups each containing n = 50 flies treated with F15 powder containing 40% pyriproxyfen.

The amount of PP on treated sterile males decreased over time after treatment (Fig. 2). There were some variations in PP amount between each batch of treated sterile males. The average PP

amount per sterile male fell from about 1,000 ng just after treatment to below half that amount in only three hours. After 24 hours, the average PP dose stabilized around 100 ng until ten

days. The average loss of PP by sterile males was 63% three hours post-treatment and 88% after 24 hours.

Persistence of pyriproxyfen (log scale) over time after treatment of sterile males of Glossina palpalis gambiensis with F15 + PP (n = 2 flies/time/batch).

All the 60 virgin females used for this experiment mated with the sterile males treated with PP. The average amounts of PP present on sterile males after mating, three and 24 hours after

treatment, were 365.5 ± 133.8 ng and 218.1 ± 73.6 ng respectively (Table 1). This difference was significant (Wilcoxon test: p