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Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Aim: The rise in rice production in the district of Malanville, Northen Benin, is a present concern, as it has resulted in the widespread usage of pesticides for crop protection. This could impact human health but also life cycle of Anopheles gambiae , the main vector of malaria., Methods: Therefore, insecticide susceptibility bioassays were carried out on populations of An. gambiae s.l aged to 3-5 days old (two from areas where insecticide is highly used and other two areas of low insecticide use) and subjected to insecticide-impregnated papers (Permethrin 0.75%; deltamethrin 0.05%; DDT 4% and bendiocarb 0.1%) following WHO protocol. Polymerase Chain Reactions (PCRs) were used for the detection of Acethlylcholinestrase (Ace-1) and the knock down resistance (kdr) L1014F mutations in An. gambiae populations. Finally, indirect bioassays were conducted for the investigating on the factors affecting the life cycle of An. gambiae due to the use of pesticides., Results: An. gambiae from the four sites were resistant to DDT (6 to 8% and 10 to 14% respectively from areas of high and low dose), pyrethroids (22 to 26% and 30 to 36% for permethrin, from areas of high and low dose respectively and 66 to 70% and 72 to 80% for deltamethrin, from high and low dose) but susceptible to carbamate. The kdr L1014F mutation was detected in An. gambiae populations (0.88 to 0.90 and 0.84 to 0.88 from high and low dose, respectively). The ace-1 was detected at low frequencies (<0.002). Bioassays on the impacts of the use of pesticides in the life cycle of An. gambiae showed that soil substrates with pesticides residues have a negative impact on the life cycle eggs of An. gambiae ., Conclusion: These findings confirmed the negative impacts of pesticides use in rice farming and its impacts on the life cycle of An. gambiae ., Competing Interests: The authors declare that there is no conflict of interest associated with this study., (© 2023 Kamuzu University of Health Sciences.)

Pyrethroid resistance is widespread in sub-Saharan Africa. The objective of this study was to assess the insecticide resistance intensity in Anopheles gambiae s.l. (Diptera: Culicidae) in four districts of Benin in order to better understand how pyrethroid-only nets are likely to be effective. Thus, adult females of An. gambiae s.l., reared from field-collected larvae were used for assessing resistance intensity to permethrin and deltamethrin. They were tested at 1×, 5×, and 10× the diagnostic dose, using both WHO susceptibility tube testing and CDC bottle bioassays. Identification of molecular species, as well as of L1014F Kdr and Ace-1R mutations was performed using the PCR. The level of expression of biochemical enzymes was also evaluated. Overall, moderate to high resistance intensity to permethrin and deltamethrin was observed, irrespective of the testing method. While the L1014F Kdr frequency was high (>75%), Ace-1R was low (≤6%) in An. gambiaes.s. and Anopheles coluzzii, the two predominant species [52% (95% CI: 44.8-59.1) and 45% (95% CI: 38.0-52.2), respectively]. Anopheles arabiensis was found at very low frequency (3%, 95%CI: 1.1-6.4). For Biochemical analyses, α and β-esterases were over-expressed in all four districts, while mixed-function oxidases (MFOs) were over-expressed in only one. Overall, the two testing methods led to comparable conclusions, though there were a few inconsistencies between them. The moderate-high resistance intensity observed in the study area suggests that dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) may provide better control of insecticide-resistant mosquitoes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study investigated in 8 villages of the Plateau region the coverage, usage, physical integrity, and bio-efficacy of the Olyset nets distributed nationwide by the Benin's National Malaria Control Programme in July 2011. The questionnaire administered as well as the observations made in the households allowed estimating the coverage and usage rates of the 2011 Olyset nets. While their physical integrity was assessed through standard WHO methodology, their bio-efficacy was evaluated through gas chromatography, and WHO cone testing performed with the Kisumu susceptible strain. Mosquito collections through human landing catches (HLCs) were also performed in torn nets to assess if a loss of protection of sleepers occurred as the nets fabric integrity got more damaged. Nine months postdistribution, the coverage and usage rates of the 2011 Olyset nets were 67.4% (95% CI: 65.8-68.9) and 73.3% (95% CI: 70.7-75.8) respectively. About 28% of the 2011 Olyset nets were torn. A drastic drop of the insecticide quantity on the fibers of the nets [from 7.08 µg (95% CI: 5.74-8.42) to 0.2 µg (95% CI: 0.01-0.38)] as well as mortality rates <80% were observed with most nets evaluated. Moreover, the biting rates of An. gambiae s.l. (Diptera: Culicidae) inside torn nets increased in line with their fabric integrity loss. These data support the conclusion that future deployment of nets in the field must be strengthened by community sensitization on their correct use in order to postpone as much as possible appearance of holes and loss of insecticidal activity and encourage repairing of torn nets., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics., Methods: Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site., Results: Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain)., Conclusion: Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control., (© 2021. The Author(s).)

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.

Background: Insecticide resistance is a growing concern for malaria control and vector control effectiveness relies on assessing it distribution and understanding its evolution., Methods: We assessed resistance levels and the frequencies of two major target-site mutations, L1014F-VGSC and G119S-ace-1, conferring resistance to pyrethroids (PYRs) and carbamates/organophosphates (CXs/OPs) insecticides. These data were compared to those acquired between 2006 and 2010 to follow resistance evolutionary trends over ten years., Results: We report the results of a 3-year survey (2013-2015) of insecticide resistance in 13 localities across the whole country of Benin. Permethrin (PYR) resistance was found in all populations tested, L1014F-VGSC being almost fixed everywhere, while bendiocarb resistance was limited to a few localities, G119S-ace-1 remaining rare, with very limited variations during surveyed period. Interestingly, we found no effect of the type of insecticide pressure on the dynamics of these mutations., Conclusions: These results confirm both the high prevalence of PYR resistance and the potential of CXs/OPs as short- to medium-term alternatives in Benin. They also underline the need for regular resistance monitoring and informed management in their usage, as the G119S-ace-1 mutation is already present in Benin and surrounding countries. Their unwise usage would rapidly lead to its spread, which would jeopardize PYR-resistant Anopheles control.

Background: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. In order to implement suitable insecticide resistance management strategies, it is necessary to understand the underlying mechanisms involved. To achieve this, the molecular basis of permethrin and DDT resistance in the principal malaria vector, Anopheles funestus from inland Benin (Kpome), was investigated., Results: Here, using a microarray-based genome-wide transcription and qRT-PCR analysis, we showed that metabolic resistance mechanisms through over-expression of cytochrome P450 and glutathione S-transferase genes (GSTs) are a major contributor to DDT and permethrin resistance in Anopheles funestus from Kpome. The GSTe2 gene was the most upregulated detoxification gene in both DDT- [fold-change (FC: 16.0)] and permethrin-resistant (FC: 18.1) mosquitoes suggesting that upregulation of this gene could contribute to DDT resistance and cross-resistance to permethrin. CYP6P9a and CYP6P9b genes that have been previously associated with pyrethroid resistance were also significantly overexpressed with FC 5.4 and 4.8, respectively, in a permethrin resistant population. Noticeably, the GSTs, GSTd1-5 and GSTd3, were more upregulated in DDT-resistant than in permethrin-resistant Anopheles funestus suggesting these genes are more implicated in DDT resistance. The absence of the L1014F or L1014S kdr mutations in the voltage-gated sodium channel gene coupled with the lack of directional selection at the gene further supported that knockdown resistance plays little role in this resistance., Conclusions: The major role played by metabolic resistance to pyrethroids in this An. funestus population in Benin suggests that using novel control tools combining the P450 synergist piperonyl butoxide (PBO), such as PBO-based bednets, could help manage the growing pyrethroid resistance in this malaria vector in Benin.

Background: The goal of the study is to investigate if local agricultural practices have an impact on malaria transmission in four villages located in the same geographical area within a radius of 15 kilometers. Among the villages, one (Itassoumba) is characterized by the presence of a large market garden and fishpond basins, the three others (Itakpako, Djohounkollé and Ko-koumolou) are characterized by traditional food-producing agriculture., Methods: Malaria transmission was evaluated using human-landing catches, both indoors and outdoors, two nights per month for 12 months. Field collected females An. gambiae s.l. were exposed for 1 hour to 0.75% permethrin and 0.05% deltamethrin using WHO insecticide susceptibility test kits and procedures. The presence of the kdr mutation was analyzed by PCR., Results: Anopheles gambiae s.s form M (93.65%), was identified as the main malaria vector. Its susceptibility level to pyrethroids was the same (p > 0.05) in all villages. kdr mutation frequencies are 81.08 in Itakpako, 85 in Itassoumba, 79.73 in Djohounkollé and 86.84 in Ko-Koumolou (p = 0.63). The entomological inoculation rate ranged from 9.62 to 21.65 infected bites of An. gambiae per human per year in Djohounkollé, Itakpako and Ko-Koumolou against 1159.62 in Itassoumba (p < 0.0001)., Conclusion: The level of resistance of An. gambiae to pyrethroids was the same in the four villages. The heterogeneous character of malaria epidemiology was confirmed. The creation of fishponds basins and the development of market-gardening activities increased drastically the malaria transmission in Itassoumba.