Interactions entre l’état physiologique du moustique et le mode d’action des répulsifs chez les vecteurs de pathogènes

Mosquito-borne diseases are a permanent public health threat. The main way to protect populations against infectious bites relies on the use of insecticides, but their efficacy is threatened by the spread of resistance mechanisms among vector populations. In this context, repellents are a promising tool for reducing both mosquito nuisance and infection risk. Among them, DEET (N, N-diéthyl-3-méthylbenzamide) has proven great efficacy both in laboratory and field experiments. Despite this, repellents, are still used from an empirical way and their mode of action remains poorly understood. The aim of the present project was to: i) participate to the evaluation of the potential of integration of the DEET repellent in mosquito control, ii) measure the impact of mosquito physiological parameters of epidemiological relevance such as experience, age and infection, on the efficacy of repellents and insecticides, and iii) quantify the impact of repellents and insecticides on mosquito life-history traits. Experiments were performed on Anopheles gambiae and Aedes albopictus, vectors of the malaria parasite and of arboviruses such as dengue, respectively. Our results allowed to demonstrate that mosquito physiological state influences the efficacy of control tools; first, after successfully obtaining a blood meal in contact with permethrin, mosquitoes carrying kdr mutation are no longer inhibited by this compound at the subsequent exposure, at the recommended dose for net impregnation and under our experimental design. On the contrary, a first blood meal obtained in presence of DEET does not affect its efficacy at the following exposure. Then, DEET appeared to be more efficient against old mosquitoes that against younger ones, and results are consistent in the two species, independently of the insecticide resistance status. Besides, efficacy of DEET and deltamethrin is not modified when mosquitoes are infected with Plasmodium falciparum. However, an increased mortality was observed in anopheles when infected with transmissible stages (i.e. sporozoites), independently of chemical exposure, which suggest a cost of infection and seems to be “replicate-dependent”. Finally, DEET produces a long-term impact on mosquito fecundity and fertility, which is not observed with permethrin under our protocol. These results give insights to redefine the priorities in mosquito control programs, in order to specifically targeting mosquitoes that are the most susceptible to transmit pathogens. These observations also underline the need for considering both mosquito physiological state and the long-term impact of repellents and insecticides during the evaluation of control tools.Les maladies transmises par les moustiques représentent une menace permanente en santé publique. La principale mesure pour protéger les populations contre les piqûres infectieuses repose actuellement sur l’utilisation d’insecticides, mais celle-ci est menacée par la propagation des mécanismes de résistance au sein des populations de moustiques. Dans ce contexte, les répulsifs représentent un outil de choix pour réduire à la fois les nuisances provoquées par les moustiques et le risque d’infection. Parmi eux, le DEET (N, N-diéthyl-3-méthylbenzamide) a prouvé une efficacité remarquable aussi bien lors d’expérimentations en laboratoire que sur le terrain. Malgré cela, les répulsifs ont toujours été utilisés de manière empirique, et leur mode d’action reste très mal connu. Le but de ce projet de thèse a été : i) de participer à l’évaluation du potentiel d’intégration du répulsif DEET dans la lutte antivectorielle, ii) de mesurer l’impact de paramètres physiologiques du moustique d’intérêt épidémiologique tels que l’expérience, l’âge et l’infection sur l’efficacité des méthodes de lutte, répulsifs et insecticides pyréthrinoïdes et iii) de quantifier l’impact des répulsifs et insecticides sur les traits d’histoire de vie des moustiques. Les expériences ont été réalisées sur Anopheles gambiae et Aedes albopictus, respectivement vecteurs de l’agent du paludisme et d’arbovirus tels que le virus de la dengue. Nos résultats ont permis de démontrer que l’état physiologique du moustique influe sur l’efficacité des méthodes de lutte ; premièrement, les moustiques porteurs de la mutation kdr ayant obtenu un repas de sang en contact avec de la perméthrine ne sont plus irrités par ce composé lors d’une seconde exposition, dans les conditions testées et à une dose recommandée pour l’imprégnation de moustiquaires. Au contraire, un repas de sang obtenu en présence DEET n’a pas affecté l’efficacité de ce composé à inhiber un repas de sang à l’exposition suivante. Ensuite, le DEET s’est montré plus efficace chez les moustiques âgés que chez les moustiques jeunes, et les résultats sont similaires chez les deux espèces et indépendants du statut de résistance aux insecticides. D’autre part, l’efficacité de la deltaméthrine et du DEET ne sont pas modifiés lorsque les moustiques sont infectés par le parasite Plasmodium falciparum. Cependant, une augmentation de la mortalité a été observée chez les femelles anophèles infectées au stade sporozoites lors de certaines expérimentations, indépendamment du traitement chimique de la moustiquaire, et suggère un coût de l’infection « réplicat-dépendant ». Enfin, le DEET génère un impact au long terme sur la fécondité et la fertilité des moustiques, effets qui ne sont pas observés dans notre protocole pour la perméthrine. Ces résultats donnent donc des pistes pour redéfinir les priorités dans les programmes de lutte, afin de cibler en premier lieu les moustiques les plus à même de transmettre des pathogènes. Ils soulignent également la nécessité de considérer à la fois l’état physiologique du moustique et l’impact sur le long terme des insecticides et répulsifs lors de l’évaluation des outils de lutte

Interactions between mosquito physiological state and repellents' mode of action in vectors of pathogens

Les maladies transmises par les moustiques représentent une menace permanente en santé publique. La principale mesure pour protéger les populations contre les piqûres infectieuses repose actuellement sur l’utilisation d’insecticides, mais celle-ci est menacée par la propagation des mécanismes de résistance au sein des populations de moustiques. Dans ce contexte, les répulsifs représentent un outil de choix pour réduire à la fois les nuisances provoquées par les moustiques et le risque d’infection. Parmi eux, le DEET (N, N-diéthyl-3-méthylbenzamide) a prouvé une efficacité remarquable aussi bien lors d’expérimentations en laboratoire que sur le terrain. Malgré cela, les répulsifs ont toujours été utilisés de manière empirique, et leur mode d’action reste très mal connu. Le but de ce projet de thèse a été : i) de participer à l’évaluation du potentiel d’intégration du répulsif DEET dans la lutte antivectorielle, ii) de mesurer l’impact de paramètres physiologiques du moustique d’intérêt épidémiologique tels que l’expérience, l’âge et l’infection sur l’efficacité des méthodes de lutte, répulsifs et insecticides pyréthrinoïdes et iii) de quantifier l’impact des répulsifs et insecticides sur les traits d’histoire de vie des moustiques. Les expériences ont été réalisées sur Anopheles gambiae et Aedes albopictus, respectivement vecteurs de l’agent du paludisme et d’arbovirus tels que le virus de la dengue. Nos résultats ont permis de démontrer que l’état physiologique du moustique influe sur l’efficacité des méthodes de lutte ; premièrement, les moustiques porteurs de la mutation kdr ayant obtenu un repas de sang en contact avec de la perméthrine ne sont plus irrités par ce composé lors d’une seconde exposition, dans les conditions testées et à une dose recommandée pour l’imprégnation de moustiquaires. Au contraire, un repas de sang obtenu en présence DEET n’a pas affecté l’efficacité de ce composé à inhiber un repas de sang à l’exposition suivante. Ensuite, le DEET s’est montré plus efficace chez les moustiques âgés que chez les moustiques jeunes, et les résultats sont similaires chez les deux espèces et indépendants du statut de résistance aux insecticides. D’autre part, l’efficacité de la deltaméthrine et du DEET ne sont pas modifiés lorsque les moustiques sont infectés par le parasite Plasmodium falciparum. Cependant, une augmentation de la mortalité a été observée chez les femelles anophèles infectées au stade sporozoites lors de certaines expérimentations, indépendamment du traitement chimique de la moustiquaire, et suggère un coût de l’infection « réplicat-dépendant ». Enfin, le DEET génère un impact au long terme sur la fécondité et la fertilité des moustiques, effets qui ne sont pas observés dans notre protocole pour la perméthrine. Ces résultats donnent donc des pistes pour redéfinir les priorités dans les programmes de lutte, afin de cibler en premier lieu les moustiques les plus à même de transmettre des pathogènes. Ils soulignent également la nécessité de considérer à la fois l’état physiologique du moustique et l’impact sur le long terme des insecticides et répulsifs lors de l’évaluation des outils de lutte.Mosquito-borne diseases are a permanent public health threat. The main way to protect populations against infectious bites relies on the use of insecticides, but their efficacy is threatened by the spread of resistance mechanisms among vector populations. In this context, repellents are a promising tool for reducing both mosquito nuisance and infection risk. Among them, DEET (N, N-diéthyl-3-méthylbenzamide) has proven great efficacy both in laboratory and field experiments. Despite this, repellents, are still used from an empirical way and their mode of action remains poorly understood. The aim of the present project was to: i) participate to the evaluation of the potential of integration of the DEET repellent in mosquito control, ii) measure the impact of mosquito physiological parameters of epidemiological relevance such as experience, age and infection, on the efficacy of repellents and insecticides, and iii) quantify the impact of repellents and insecticides on mosquito life-history traits. Experiments were performed on Anopheles gambiae and Aedes albopictus, vectors of the malaria parasite and of arboviruses such as dengue, respectively. Our results allowed to demonstrate that mosquito physiological state influences the efficacy of control tools; first, after successfully obtaining a blood meal in contact with permethrin, mosquitoes carrying kdr mutation are no longer inhibited by this compound at the subsequent exposure, at the recommended dose for net impregnation and under our experimental design. On the contrary, a first blood meal obtained in presence of DEET does not affect its efficacy at the following exposure. Then, DEET appeared to be more efficient against old mosquitoes that against younger ones, and results are consistent in the two species, independently of the insecticide resistance status. Besides, efficacy of DEET and deltamethrin is not modified when mosquitoes are infected with Plasmodium falciparum. However, an increased mortality was observed in anopheles when infected with transmissible stages (i.e. sporozoites), independently of chemical exposure, which suggest a cost of infection and seems to be “replicate-dependent”. Finally, DEET produces a long-term impact on mosquito fecundity and fertility, which is not observed with permethrin under our protocol. These results give insights to redefine the priorities in mosquito control programs, in order to specifically targeting mosquitoes that are the most susceptible to transmit pathogens. These observations also underline the need for considering both mosquito physiological state and the long-term impact of repellents and insecticides during the evaluation of control tools

Semiochemical oviposition cues to control Aedes aegypti gravid females: state of the art and proposed framework for their validation.

peer reviewedIn the fight against mosquito-borne diseases, odour-based lures targeting gravid females represent a promising alternative to conventional tools for both reducing mosquito populations and monitoring pathogen transmission. To be sustainable and effective, they are expected to use semiochemicals that act specifically against the targeted vector species. In control programmes directed against Aedes aegypti, several candidates of different origins (conspecifics, plants) have already been identified as potential oviposition attractants or repellents in laboratory experiments. However, few of these candidates have received validation in field experiments, studies depicting the active molecules and their mode of perception are still scarce, and there are several methodological challenges (i.e. lack of standardization, differences in oviposition index interpretation and use) that should be addressed to ensure a better reproducibility and accelerate the validation of candidates. In this review, we address the state of the art of the compounds identified as potential candidates for trap development against Ae. aegypti and their level of validation. We also offer a critical methodological analysis, highlight remaining gaps and research priorities, and propose a workflow to validate these candidates and to increase the panel of odours available to specifically trap Ae. aegypti

Prior contact with permethrin decreases its irritancy at the following exposure among a pyrethroid-resistant malaria vector Anopheles gambiae

International audienceInsecticide-treated nets (ItNs) remain major components for vector control despite the spread of resistance mechanisms among mosquito populations. Multiple exposures to pyrethroids may induce physiological and behavioral changes in mosquitoes, possibly reducing efficacy of control tools. Despite epidemiological relevance, the effects of multiple exposures to pyrethroids on their efficacy against pyrethroid-resistant mosquitoes has received little interest. In the present study, we assessed the effects of a blood-meal successfully obtained upon a permethrin-treated net on the success at taking a second blood-meal in presence of permethrin in Anopheles gambiae, carrying pyrethroid resistance alleles. We also measured the impact of exposure to permethrin on life-history traits to address the delayed efficacy of ITNs. Our results showed that females that successfully blood-fed upon a permethrin-treated net were no longer inhibited by permethrin at the following exposure. Blood-meal inhibition due to permethrin was not affected by female size nor by exposure of mothers when testing the offspring, allowing to discard the effect of genetic or physiological selection. Besides, in our assays, exposure to permethrin did not affect mosquito fecundity, fertility nor survival. These results give insights to understand the long-term efficacy of ITNs, and allow to reevaluate the criteria used when choosing compounds for fighting malaria mosquitoes

Effect of DEET-multiple exposures on behavior and life history traits in the malaria mosquito Anopheles gambiae (s.s.)

International audienceBackground: Vector-borne diseases are major public health concerns, and their control is threatened by the spread of insecticide resistance in vector populations. In this context, the use of repellents is an alternative approach to limit vector-host interactions. However, prior exposure to repellents is suspected to affect mosquito behavior at the subsequent exposure, possibly reducing the efficacy of the compound. Despite this, the effect of mosquito experience on repellent efficacy remains poorly documented. In the present study, we tested whether a first blood meal successfully obtained upon a DEET-treated net would affect the success at taking a second blood meal in spite of DEET in the malaria mosquito Anopheles gambiae (s.s.). The impact of DEET on mosquito life history traits after the first and the second exposure was also measured, in order to assess the long-term consequences of multiple exposures to DEET in vector insects.Results: A first blood meal obtained upon a DEET-treated net did not influence the success of An. gambiae females to take a second blood meal in spite of DEET. However, data showed that a prior exposure to DEET negatively affected all life history traits tested in this study related to fecundity and fertility. DEET pre-exposed females displayed a reduction in blood engorgement at the second exposure, as well as a reduction in the number of eggs laid and in the proportion of offspring that reach adult stage. Also, an increase of mosquito activity was observed during the second blood meal in DEET-pre-exposed females. Taken together, these data suggest an overall impact of DEET exposure on mosquito fitness.Conclusions: Our results did not evidence any effect of a prior exposure to DEET on its efficacy during the second exposure. However, data show a negative impact of DEET exposure on mosquito fitness. These results give insights to understand the long-term efficacy of the most used mosquito repellent, and highlight that DEET induces deleterious effects on mosquito fitness in addition to repellency, potentially increasing its efficacy for controlling vector-borne diseases

Behavioural and antennal responses of Aedes aegypti (l.) (Diptera: Culicidae) gravid females to chemical cues from conspecific larvae.

Mass trapping of gravid females represents one promising strategy for the development of sustainable tools against Aedes aegypti. However, this technique requires the development of effective odorant lures that can compete with natural breeding sites. The presence of conspecific larvae has been shown to stimulate oviposition. Hence, we evaluated the role of four major molecules previously identified from Ae. aegypti larvae (isovaleric, myristoleic, myristic [i.e. tetradecanoic], and pentadecanoic acids) on the oviposition of conspecific females, as well as their olfactory perception to evaluate their range of detection. Using flight cage assays, the preference of gravid females to oviposit in water that previously contained larvae (LHW) or containing the four larval compounds was evaluated. Then, compounds and doses inducing the highest stimulation were challenged for their efficacy against LHW. Only isovaleric acid elicited antennal response, suggesting that the other compounds may act as taste cues. Pentadecanoic acid induced significant oviposition stimulation, especially when dosed at 10 ppm. Myristoleic acid and isovaleric acid deterred oviposition at 10 and 100 ppm, while no effect on oviposition was observed with myristic acid irrespectively of the dose tested. When the four compounds were pooled to mimic larvae's chemical signature, they favored oviposition at 1 ppm but negatively affected egg-laying at higher concentrations. When properly dosed, pentadecanoic acid and the blend of compounds may be promising lures for ovitraps as they could compete with LHW. Due to their low volatility, their effect should be further evaluated under field conditions, in addition with long-range attractants for developing effective tools against gravid females

DEET efficacy increases with age in the vector mosquitoes Anopheles gambiae s.s. and Aedes albopictus (Diptera: Culicidae)

International audienceOld mosquitoes are the most likely to transmit pathogens due to the higher probability that they will be exposed to pathogens, and the time required before a mosquito becomes infectious, the extrinsic incubation period (EIP). However, old mosquitoes are rarely considered in the evaluation of control tools. This study evaluated the effect of mosquito aging on the repellent efficacy of N,N-diethyl-3-methylbenzamide (DEET) in two vector mosquitoes, Anopheles gambiae s.s. (Giles) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae), respective vectors of malaria parasites and arboviruses such as dengue, chikungunya, and Zika viruses. For both mosquito species, DEET-induced inhibition of blood-feeding was assessed in three age classes. Life-history traits related to mosquito fecundity and survival following DEET exposure were also measured. Results showed that, in both species, bloodmeal inhibition induced by DEET was significantly higher in old females (>18 d old) than in younger ones (<13 d old). Life history traits recording showed no combined effects of DEET and aging on mosquito blood engorgement, oviposition rate, number of eggs laid nor survival; however, age effects are reported for all these traits. These results highlight the need for taking into account mosquito age in the evaluation of integrated mosquito management tools. They also suggest that the control of vector-borne pathogens with a long EIP could be improved by targeting old mosquitoes and supports the efficacy of repellents in the fight against mosquito-borne diseases

Efficacy of vector control tools against malaria-infected mosquitoes

International audienceWithin mosquito vector populations, infectious mosquitoes are the ones completing the transmission of pathogens to susceptible hosts and they are, consequently, of great epidemiological interest. Mosquito infection by malaria parasites has been shown to affect several traits of mosquito physiology and behavior, and could interplay with the efficacy of control tools. In this study, we evaluated, in pyrethroid resistant Anopheles gambiae, the effect of mosquito infection with the human malaria parasite Plasmodium falciparum on the efficacy of nets treated with either the insecticide deltamethrin or the repellent DEET, measuring (i) mosquito success to pass through the net, (ii) blood-feeding on a host and (iii) chemicals-induced mortality. Infection of mosquitoes at non-infectious stage did not affect their success to pass through the net, to blood-feed, nor chemicals-induced mortality. At infectious stage, depending on replicates, infected mosquitoes had higher mortality rates than uninfected mosquitoes, with stronger effect in presence of DEET. This data evidenced a cost of infection on mosquito survival at transmissible stages of infection, which could have significant consequences for both malaria epidemiology and vector control. This stresses the need for understanding the combined effects of insecticide resistance and infection on the efficacy on control tools. Mosquito-borne diseases are considerable public health issues, mostly affecting populations in developing countries 1,2. To reduce their incidence, controlling vector mosquitoes and limiting their contact with human hosts remains the most effective strategy 3. One promising approach for achieving this goal would be to selectively target, within mosquito populations, individuals that are the most dangerous for humans 4. Particularly, mosquitoes carrying transmissible forms of pathogens in their salivary glands (i.e. infectious mosquitoes), are of great epidemiological relevance. Yet, the implementation of control means that could specifically reach infectious mosquitoes is dependent upon an extensive knowledge of the effects of mosquito infection on its behavior and physiology. Infection by pathogens has been shown to affect mosquito phenotypic traits 5. Malaria-infected Anopheles mosquitoes generally display increase attraction, biting and feeding rate 6-10. Interestingly, these changes are often concomitant with the presence of transmissible stages of the pathogen and may contribute to increase the number of contacts between human hosts and infectious vectors 11. These effects may lead to substantial epidemiological consequences, with transmission rates of mosquito borne pathogens being potentially much higher than expected 12. On the other hand, infection by malaria parasites may induce fitness costs on mosquitoes, reducing their survival. This can be especially true when infection is associated with other biotic and abiotic stresses 13-15. Consequently, all the behavioral and physiological changes associated with infection could directly impact the efficacy of control means. To date, conventional control tools mostly rely on the use of insecticide-treated nets (ITNs), although their efficacy could be threatened by the increase of insecticide resistance mechanisms in mosquito populations 16. Besides, repellents such as DEET (N, N-diéthyl-3-méthylbenzamide) offer a great potential both in cutaneou

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies