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.

Background: In May 2019, a confirmed dengue fever case was detected at the local hospital of Abomey-Calavi Commune in southern Benin. In Benin, there remains a dearth of literature concerning the distribution and biology of Aedes aegypti, the principal vector of dengue fever. This study was initiated by the Ministry of Health to partially fill this gap. The findings allowed us to assess the arboviral transmission risk incurred by the population of Abomey-Calavi to support programmatic decision-making. Methods: Entomological assessments were conducted in 5% of the houses, meaning 314 houses selected from 11 boroughs in Abomey-Calavi Centre district and 9 villages in Hêvié district. The surveyed breeding sites were water containers located in (domestic) and around (peri-domestic) the dwellings. When a container was positive (housing larvae), a portion of the immature population was sampled with a larval dipper and poured into labeled jars. Immatures were then reared to adulthood at the Centre de Recheche Entomologique de Cotonou (CREC) insectary. Adult mosquitoes were morphologically identified to species level by site and, a subsample of the collected Ae. aegypti mosquitoes were used for WHO susceptibility tube tests. Results: Of the 1372 adult Aedes specimens which emerged from the collected larvae and pupae, 1356 Ae. aegypti (98.83%), 10 Ae. luteocephalus, and 4 Ae. vittatus were identified. The Breteau indices were 160.2 in Abomey-Calavi Centre and 150 in Hêvié, whereas the House indices were 58.5% and 61.6% in the respective districts. WHO insecticide susceptibility tube tests showed that the mortality rates were 38.71% in Abomey-Calavi Centre and 85.71% in Hêvié for permethrin, and 72.22% in Abomey-Calavi Centre and 100% in Hêvié for deltamethrin. Conclusion: The two districts were highly infested by Ae. aegypti whose breeding sites were mostly man-made. Considering this, human behavioral change to substantially reduce the number of larval habitats is necessary to control the vector populations. As Aedes mosquitoes are day biters, the use of repellents such as ointments and smoke coils can also be useful.

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. Graphical

The main objective of the present study is to assess the preferences in terms of vertebrate hosts of Anopheles coluzzii, the main malaria vector in the pastoral area of Malanville, Benin, where rice cultivation and livestock are the main source of income for the populations. Adult mosquitoes were collected through pyrethrum spray catch, and human landing catch in two communes in Benin: Malanville, a pastoral area, and Porto-Novo, a nonpastoral area. Molecular species identification was performed through PCR within the Anopheles gambiae complex. Blood meal origin and P. falciparum sporozoite infection were determined using ELISA blood meal and circumsporozoite protein tests, respectively. Overall, 97% of females of An. gambiae s.l. were An. coluzzii, with biting behavior more pronounced outdoors in the pastoral area. In Malanville, the main vertebrate hosts on which females An. coluzzii blood fed were goats (44%), humans (24.29%), bovines (22%), and pigs (1.4%). Our results also showed that single-host blood meals (human: 24.29% or animal: 68%) were mostly observed compared to mixed blood meals (8.58%). The human biting rate (HBR) and P. falciparum sporozoite rate (SR) of An. coluzzii were 66.25 bites/man/night and 0.77%, respectively. However, in the nonpastoral zone (Porto-novo), 93.98% of samples were An. coluzzii. The latter blood-fed mostly (86.84%) on humans, with an estimated HBR of 21.53 b/m/n and SR of 5.81%. The present study revealed an opportunistic and zoophagic behavior of An. coluzzii in the Malanville area with an overall low mean SR.

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-1^R 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-1^R 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 efficacy of a vector control tool in reducing mosquito biting is crucial for its acceptability. The present study compared the vector density of Culex spp. And Mansonia spp. across clusters, which received two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) and a standard pyrethroid-only LLIN, and assessed the seasonality of these mosquito genera. A total of 85,723 Culex spp. and 144,025 Mansonia spp. were caught over the study period. The density of Culex and Mansonia was reduced in all three arms over the study period. There was no evidence of a significant reduction in the indoor or outdoor density of Culex spp. in either dual-a.i. LLIN arm as compared to the standard pyrethroid-only net arm. A similar trend was observed with Mansonia spp. A high density of Culex spp. was found both in rainy and dry seasons, while for Mansonia spp., this was mainly observed during the rainy season. These results suggest that the novel insecticides in the dual-a.i. LLINs did not have an additional impact on these species and that pyrethroids might still be effective on them. Further work is required to determine whether these species of mosquitoes have resistance to the insecticides tested in this trial.