Entomological assessment of hessian fabric transfluthrin vapour emanators for protecting against outdoor-biting Aedes aegypti in coastal Tanzania.

BACKGROUND: A low technology emanator device for slowly releasing vapour of the volatile pyrethroid transfluthrin was recently developed in Tanzania that provides robust protection against night biting Anopheles and Culex vectors of malaria and filariasis for several months. Here these same emanator devices were assessed in Dar es Salaam city, as a means of protection against outdoor-biting Aedes (Stegomia) aegypti, the most important vector of human arboviruses worldwide, in parallel with similar studies in Haiti and Brazil. METHODS: A series of entomological experiments were conducted under field and semi-field conditions, to evaluate whether transfluthrin emanators protect against wild Ae. aegypti, and also compare the transfluthrin responsiveness of Ae. aegypti originating from wild-caught eggs to established pyrethroid-susceptible Ae. aegypti and Anopheles gambiae colonies. Preliminary measurements of transfluthrin vapour concentration in air samples collected near treated emanators were conducted by gas chromatography-mass spectrometry. RESULTS: Two full field experiments with four different emanator designs and three different transfluthrin formulations consistently indicated negligible reduction of human landing rates by wild Ae. aegypti. Under semi-field conditions in large cages, 50 to 60% reductions of landing rates were observed, regardless of which transfluthrin dose, capture method, emanator placement position, or source of mosquitoes (mildly pyrethroid resistant wild caught Ae. aegypti or pyrethroid-susceptible colonies of Ae. aegypti and An. gambiae) was used. Air samples collected immediately downwind from an emanator treated with the highest transfluthrin dose (15g), contained 12 to 19 µg/m3 transfluthrin vapour. CONCLUSIONS: It appears unlikely that the moderate levels of pyrethroid resistance observed in wild Ae. aegypti can explain the modest-to-undetectable levels of protection exhibited. While potential inhalation exposure could be of concern for the highest (15g) dose evaluated, 3g of transfluthrin appears sufficient to achieve the modest levels of protection that were demonstrated entomologically. While the generally low levels of protection against Aedes reported here from Tanzania, and from similar entomological studies in Haiti and Brazil, are discouraging, complementary social science studies in Haiti and Brazil suggest end-users perceive valuable levels of protection against mosquitoes. It therefore remains unclear whether transfluthrin emanators have potential for protecting against Aedes vectors of important human arboviruses.

Entomological assessment of hessian fabric transfluthrin vapour emanators as a means to protect against outdoor-biting Aedes after providing them to households for routine use in Port-au-Prince, Haiti.

BACKGROUND: A simple treated fabric device for passively emanating the volatile pyrethroid transfluthrin was recently developed in Tanzania that protected against nocturnal Anopheles and Culex mosquitoes for several months. Here these transfluthrin emanators were assessed in Port-au-Prince, Haiti against outdoor-biting Aedes. METHODS: Transfluthrin emanators were distributed to participating households in poor-to-middle class urban neighbourhoods and evaluated once every two months in terms of their effects on human landing rates of wild Aedes populations. A series of three such entomological assessment experiments were conducted, to examine the influence of changing weather conditions, various transfluthrin formulations and emanator placement on protective efficacy measurements. Laboratory experiments assessed resistance of local Aedes aegypti to transfluthrin and deltamethrin, and the irritancy and repellency of the transfluthrin-treated fabric used in the field. RESULTS: Across all three entomological field assessments, little evidence of protection against wild Ae. aegypti was observed, regardless of weather conditions, transfluthrin formulation or emanator placement: A generalized linear mixed model fitted to the pooled data from all three assessment rounds (921 females caught over 5129 hours) estimated a relative landing rate [95% Confidence interval] of 0.87 [0.73, 1.04] for users of treated versus untreated emanators (P = 0.1241). Wild Ae. aegypti in this setting were clearly resistant to transfluthrin when compared to a fully susceptible colony. CONCLUSIONS: Transfluthrin emanators had little if any apparent effect upon Aedes landing rates by wild Ae. aegypti in urban Haiti, and similar results have been obtained by comparable studies in Tanzania, Brazil and Peru. In stark contrast, however, parallel sociological assessments of perspectives among these same end-users in urban Haitian communities indicate strong satisfaction in terms of perceived protection against mosquitoes. It remains unclear why the results obtained from these complementary entomological and sociological assessments in Haiti differ so much, as do those from a similar set of studies in Brazil. It is encouraging, however, that similar contrasts between the entomological and epidemiological results of a recent large-scale assessment of another transfluthrin emanator product in Peru, which indicate they provide useful protection against Aedes-borne arboviral infections, despite apparently providing only modest protection against Aedes mosquito bites.

Deltamethrin and transfluthrin select for distinct transcriptomic responses in the malaria vector Anopheles gambiae.

BACKGROUND: The widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq. METHODS: A field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide. RESULTS: A rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae. CONCLUSION: This study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control.

Exposure of Anopheles gambiae larvae to a sub-lethal dose of an agrochemical mixture induces tolerance to adulticides used in vector control management.

The heavy use of pesticides in agricultural areas often leads to the contamination of nearby mosquito larvae breeding sites. Exposure to complex mixtures of agrochemicals can affect the insecticide sensitivity of mosquito larvae. Our study objective was to determine whether agrochemical residues in Anopheline larval breeding sites can affect the tolerance of adults to commonly used adulticides. We focussed on Fludora® Fusion, a vector control insecticide formulation combining two insecticides (deltamethrin and clothianidin) with different modes of action. An. gambiae larvae were exposed to a sub-lethal dose of a mixture of agrochemical pesticides used in a highly active agricultural area on the Ivory Coast. Comparative bioassays with Fludora Fusion mixture and its two insecticide components (deltamethrin and clothianidin) were carried out between adult mosquitoes exposed or not to the agrochemicals at the larval stage. A transcriptomic analysis using RNA sequencing was then performed on larvae and adults to study the molecular mechanisms underlying the phenotypic changes observed. Bioassays revealed a significantly increased tolerance of adult females to clothianidin (2.5-fold) and Fludora Fusion mixture (2.2-fold) following larval exposure to agrochemicals. Significantly increased tolerance to deltamethrin was not observed suggesting that insecticide exposure affects the adult efficacy of the Fludora Fusion mixture mainly through mechanisms acting on clothianidin. Transcriptomic analysis revealed the potential of agrochemicals to induce various resistance mechanisms including cuticle proteins, detoxification action and altered insecticide sequestration. These results suggest that although the Fludora Fusion mixture is effective for adult vector control, its efficacy may be locally affected by the ecological context. The present study also suggests that, although the complex interactions between the use of agrochemicals and vector control insecticides are difficult to decipher in the field, they still must be considered in the context of insecticide resistance management programmes.

Experimental evolution supports the potential of neonicotinoid-pyrethroid combination for managing insecticide resistance in malaria vectors.

The introduction of neonicotinoids for managing insecticide resistance in mosquitoes is of high interest as they interact with a biochemical target not previously used in public health. In this concern, Bayer developed a combination of the neonicotinoid clothianidin and the pyrethroid deltamethrin (brand name Fludora Fusion) as a new vector control tool. Although this combination proved to be efficient against pyrethroid-resistant mosquitoes, its ability to prevent the selection of pyrethroid and neonicotinoid resistance alleles was not investigated. In this context, the objective of this work was to study the dynamics and the molecular mechanisms of resistance of An. gambiae to the separated or combined components of this combination. A field-derived An. gambiae line carrying resistance alleles to multiple insecticides at low frequencies was used as a starting for 33 successive generations of controlled selection. Resistance levels to each insecticide and target site mutation frequencies were monitored throughout the selection process. Cross resistance to other public health insecticides were also investigated. RNA-seq was used to compare gene transcription variations and polymorphisms across all lines. This study confirmed the potential of this insecticide combination to impair the selection of resistance as compared to its two separated components. Deltamethrin selection led to the rapid enrichment of the kdr L1014F target-site mutation. Clothianidin selection led to the over-transcription of multiple cytochrome P450s including some showing high homology with those conferring neonicotinoid resistance in other insects. A strong selection signature associated with clothianidin selection was also observed on a P450 gene cluster previously associated with resistance. Within this cluster, the gene CYP6M1 showed the highest selection signature together with a transcription profile supporting a role in clothianidin resistance. Modelling the impact of point mutations selected by clothianidin on CYP6M1 protein structure showed that selection retained a protein variant with a modified active site potentially enhancing clothianidin metabolism. In the context of the recent deployment of neonicotinoids for mosquito control and their frequent usage in agriculture, the present study highlights the benefit of combining them with other insecticides for preventing the selection of resistance and sustaining vector control activities.