The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin.

BACKGROUND: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. METHOD: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy-Weinberg equilibrium and genetic differentiation within and between populations were assessed. RESULTS: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. CONCLUSIONS: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.

Distribution and Abundance of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Benin, West Africa.

Updated information on the distribution and abundance of Aedes aegypti and Aedes albopictus is crucial to prepare African countries, such as Benin, for possible arboviral disease outbreaks. This study aims to evaluate the geographical distribution, abundance and biting behaviour of these two vectors in Benin. Three sampling techniques were used in this study. The collection of Aedes spp. adults were made through human landing catch (HLC), immatures were captured with the use of ovitraps, and a dipping technique was used for the collection of Aedes spp. in 23 communes located along the North-South and East-West transect of Benin. Adult Aedes mosquitoes were collected indoors and outdoors using HLC. Mosquito eggs, larvae and pupae were collected from containers and ovitraps. The adult mosquitoes were morphologically identified, then confirmed using a polymerase chain reaction (PCR). Overall, 12,424 adult specimens of Aedes spp. were collected, out of which 76.53% (n = 9508) and 19.32% (n = 2400) were morphologically identified as Ae. aegypti and Ae. albopictus, respectively. Geographically, Ae. aegypti was found across the North-South transect unlike Ae. albopictus, which was only encountered in the southern part of the country, with a great preponderance in Avrankou. Furthermore, an exophagic behaviour was observed in both vectors. This updated distribution of Aedes mosquito species in Benin will help to accurately identify areas that are at risk of arboviral diseases and better plan for future vector control interventions.

What can be learned from the residual efficacy of three formulations of insecticides (pirimiphos-methyl, clothianidin and deltamethrin mixture, and clothianidin alone) in large-scale in community trial in North Benin, West Africa?

BACKGROUND: In Alibori and Donga, two departments of high malaria incidence of Northern Benin, pirimiphos-methyl, mixture deltamethrin + clothianidin, as well as clothianidin were used at large scale for IRS. The present study aimed to assess the residual efficacy of these products. METHODS: Immatures of Anopheles gambiae sensu lato (s.l.) collected in the communes of Kandi and Gogounou (Department of Alibori), Djougou and Copargo (Department of Donga) were reared until adulthood. Females aged 2-5 days were used for susceptibility tube tests following the WHO protocol. The tests were conducted with deltamethrin (0.05%), bendiocarb (0.1%), pirimiphos-methyl (0.25%) and clothianidin (2% weight per volume). For cone tests performed on cement and mud walls, the An. gambiae Kisumu susceptible strain was used. After the quality control of the IRS performed 1-week post-campaign, the evaluation of the residual activity of the different tested insecticides/mixture of insecticides was conducted on a monthly basis. RESULTS: Over the three study years, deltamethrin resistance was observed in all the communes. With bendiocarb, resistance or possible resistance was observed. In 2019 and 2020, full susceptibility to pirimiphos-methyl was observed, while possible resistance to the same product was detected in 2021 in Djougou, Gogounou and Kandi. With clothianidin, full susceptibility was observed 4-6 days post-exposure. The residual activity lasted 4-5 months for pirimiphos-methyl, and 8-10 months for clothianidin and the mixture deltamethrin + clothianidin. A slightly better efficacy of the different tested products was observed on cement walls compared to the mud walls. CONCLUSION: Overall, An. gambiae s.l. was fully susceptible to clothianidin, while resistance/possible resistance was observed the other tested insecticides. In addition, clothianidin-based insecticides showed a better residual activity compared to pirimiphos-methyl, showing thus their ability to provide an improved and prolonged control of pyrethroid resistant vectors.

Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin.

BACKGROUND: Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. METHODS: Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin's insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. RESULTS: An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16-64%) and the greatest in Bohicon (12-93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44-75%) and the greatest in Bohicon (22-72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75-100%), while the ace-1 resistance allele (G119S) frequency was low (0-3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. CONCLUSION: There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin.

Efficacy of Actellic 300 CS-based indoor residual spraying on key entomological indicators of malaria transmission in Alibori and Donga, two regions of northern Benin.

BACKGROUND: The current study shows the results of three years of IRS entomological monitoring (2016, before intervention; 2017 and 2018, after intervention) performed in Alibori and Donga, northern Benin. METHODS: Mosquito collections were performed on a monthly basis using human landing catches and pyrethrum spray catches in six districts including four treated with Actellic 300 CS (Kandi, Gogounou, Djougou and Copargo) and two untreated (Bembèrèkè and Kouandé) which served as control sites. Key transmission indicators of Anopheles gambiae (s.l.) as well as the residual activity of Actellic 300 CS assessed through WHO cone tests, were determined. RESULTS: The residual efficacy duration of Actellic 300 CS after the two IRS campaigns (2017 and 2018) was 4-5 months (May-September). The parity rate and the sporozoite index of An. gambiae (s.l.) were 36.62% and 0.71%, respectively, after the first spray round in treated areas compared to 57.24% and 3.7%, respectively, in the control areas (P < 0.0001). The same trend was observed after the second spray round. After the first spray round, each person received 1.6 infective bites/month (ib/m) in the treated areas against 12.11 ib/m in the control areas, resulting in a reduction rate of 86.78%. Similarly, the entomological inoculation rate was 1.5 ib/m after the second spray round in the treated areas vs 9.75 ib/m in the control areas, corresponding to a reduction of 84.61%. A decrease in the parity rate (46.26%), sporozoite index (85.75%) and EIR (87.27%) was observed for An. gambiae (s.l.) after the first round of IRS (June-October 2017) compared to the pre-intervention period (June-October 2016). The density of An. gambiae (s.l.) ranged between 0.38-0.48 per house in treated areas vs 1.53-1.76 An. gambiae (s.l.) per house respectively after the first and second IRS rounds. CONCLUSIONS: This study showed the positive impact of IRS in reducing key entomological parameters of malaria transmission in Alibori and Donga. However, the considerable blood-feeding rate of An. gambiae (s.l.) in spray areas, stress the need for the population to sleep under long-lasting insecticidal nets (LLINs) in addition, to prevent from mosquito bites which did not succeed in resting on sprayed walls.

Insecticide resistance status, frequency of L1014F Kdr and G119S Ace-1 mutations, and expression of detoxification enzymes in Anopheles gambiae (s.l.) in two regions of northern Benin in preparation for indoor residual spraying.

BACKGROUND: This study aims to provide baseline data on the resistance status to insecticides, the frequency of mechanisms involved and the impact of the association with the synergist piperonyl butoxide (PBO) on resistant Anopheles gambiae (s.l.) populations in two regions of northern Benin, prior to an indoor residual spraying campaign and introduction of next generation long-lasting insecticidal nets (LLINs) incorporating PBO. METHODS: Adult Anopheles gambiae (s.l.) originating from larvae collected in two study regions (Alibori within the Kandi-Gogounou-Segbana districts and Donga within the Djougou-Copargo-Ouake districts) were tested with impregnated papers (bendiocarb 0.1%, pirimiphos-methyl 0.25%, permethrin 0.75% and deltamethrin 0.05%). The synergist PBO was used to check for the involvement of detoxification enzymes in pyrethroid resistant populations. Molecular analyses were performed for the identification of species within the Anopheles gambiae (s.l.) complex and kdr L1014F and G119S Ace-1 mutations. Biochemical assays assessed the activity of detoxification enzymes. RESULTS: Anopheles gambiae (s.l.) was resistant to pyrethroids, with a mortality range of 25-83% with deltamethrin and 6-55% with permethrin. A significant increase in mortality was observed after pre-exposure to PBO for both deltamethrin (63-99%) and permethrin (56-99%). With bendiocarb, An. gambiae (s.l.) were susceptible in Kandi (99% mortality), with possible resistance (92-95%) recorded in Djougou, Copargo, Gogounou, Ouake and Segbana. All study populations were fully susceptible to pirimiphos-methyl. The frequencies of resistant mutations varied according to species and sites: 0.67-0.88 for L1014F kdr and 0-0.06 for G119S Ace-1. Three study locations (Djougou, Gogounou and Kandi) showed high oxidase activity and four sites (Djougou, Ouake, Copargo and Kandi) showed elevated esterase activity. CONCLUSIONS: This study confirms resistance to pyrethroids and suggests emerging bendiocarb resistance in An. gambiae (s.l.) populations in northern Benin. However, recovery of susceptibility to pyrethroids after PBO exposure, and susceptibility to organophosphates in the An. gambiae (s.l.) populations indicate that next generation LLINs incorporating PBO synergist combined with an indoor residual spraying (IRS) campaign with organophosphate insecticides may be regarded as alternative control tools.