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Background Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. Methods Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0Asaia-positive females. Results A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). Conclusions This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa. Graphical abstract

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7, p &lt, 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1, 0.01) and PermaNet 3.0 (side) (OR = 30.1, 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

Introduction: The colours of flowers are a result of secondary metabolites that have long been used in the medical and textile industries, and those that are halochromic are used in colour display because they change color according to pH changes, but many species are yet to be studied in detail. Objective: To explore the halochromic properties and the antimicrobial potentials of the crude extracts of several ornamental plants. Methods: We used aqueous and organic solvents to extract pigments from petals of five fascinating flowers planted around International Institute of Tropical Agriculture station, Cotonou, Benin: Allamanda blanchetii, Cascabela thevetia, Eichhornia crassipes, Ixora casei and Thunbergia erecta, followed by an investigation into their halochromic properties. Antibacterial potentials of the extracts were tested on important rice pathogens: Xanthomonas oryzae pv. oryzae, and Pantoea agglomerans, which are gram-negative bacteria; and on Bacillus subtilis, a gram-positive bacterium. Results: The crude extracts of T. erecta and A. blanchetii have good halochromic properties within pH 2 – 12, exhibiting distinct colours. The chromophores of the C. thevetia, E. crassipes, and I. casei are not halochromic as the colours of the crude extracts remain the same at the pH range except pH 12 which is similar for the five extracts. Crude extracts of T. erectainhibited growth of P. agglomerans without development of resistance, whereas the bacteria developed resistance against Penicillin after 18h of incubation. T. erecta and A. blanchetii were able to inhibit growth of X. oryzae and both inhibited B. subtilis. Conclusion: Pigments from both T. erecta and A. blanchetii are good pH indicators; however, T. erecta is a better antibacterial agent than A. blanchetii because it has broad-spectrum activities against bacteria. Resumen “Propiedades halocrómicas y potenciales antimicrobianos de extractos crudos de cinco plantas ornamentales”. Introducción:Los colores de las flores son el resultado de metabolitos secundarios, que se han utilizado durante mucho tiempo en las industrias médica y textil, y los que son halocrómicos se usan en la visualización a color porque cambian de color según los cambios de pH, pero muchas especies aún no se han estudiado en detalle. Objetivo: Explorar las propiedades halocrómicas y los potenciales antimicrobianos de los extractos crudos de plantas ornamentales. Métodos: Utilizamos disolventes acuosos y orgánicos para extraer pigmentos de pétalos de cinco flores fascinantes plantadas alrededor de la Estación Internacional de Agricultura Tropical, Cotonou, Benin: Allamanda blanchetii,Cascabela thevetia, Eichhornia crassipes, Ixora casei y Thunbergia erecta, seguidas de una investigación de sus propiedades halocromáticas. Los potenciales antibacterianos de los extractos se probaron en importantes patógenos del arroz: Xanthomonas oryzae pv. oryzae, Pantoea agglomeran que son bacterias gram-negativas y en Bacillus subtilis una bacteria gram-positiva. Resultados: Los extractos crudos de T. erecta y A. blanchetii tienen buenas propiedades halocrómicas dentro del pH 2 - 12, mostrando colores distintos. Los cromóforos de C. thevetia, E. crassipes. I. casei no son halocrómicos ya que los colores de los extractos crudos permanecen iguales en el rango de pH, excepto el pH 12, que es similar para los cinco extractos. Los extractos crudos de T. erecta inhibieron el crecimiento de P. agglomerans sin desarrollo de resistencia, mientras que las bacterias desarrollaron resistencia contra la penicilina después de 18 horas de incubación. T. erecta y A. blanchetiipudieron inhibir el crecimiento de X. oryzae y ambos inhibieron B. subtilis. Conclusión:Los pigmentos de T. erecta y A. blanchetii son buenos como indicadores de pH. Sin embargo, T. erecta es un mejor agente antibacteriano que A. blanchetii ya que tiene actividades de amplio espectro contra las bacterias.

Lambda-cyhalothrin (&lambda, cyhalothrin) is the most commonly used pyrethroid insecticide for vegetable farming in Benin. This insecticide is misused and overused by farmers, and hence may pose health hazards to consumers. We monitored &lambda, cyhalothrin residues in lettuce and cabbage from farms at the market gates in Cotonou and Parakou using high performance liquid chromatography (HPLC) analysis techniques. These residues were also monitored on samples directly from farms (on-farm sampling) for 14 days post-treatment. Potential factors such as photolysis and hydrolysis involved in &lambda, cyhalothrin degradation were also screened. Results revealed that the level of &lambda, cyhalothrin residue concentrations in lettuce from Houeyiho decreased from 4.2 mg/kg on Day 1 to about 0.2 mg/kg on Day 7. On Day 9, analyzed lettuces were all &lambda, cyhalothrin free. In contrast, even 14 days after treatment of cabbage from Bawera (Parakou), we still recorded the presence of &lambda, cyhalothrin residues in analyzed samples. For samples from market gates, &lambda, cyhalothrin residues were found in lettuce from two markets out of the nine surveyed in Cotonou. Interestingly, none of these contaminated samples had residues above the maximum residue limit for lettuce (MRL = 0.5 mg/kg). Similarly, in Parakou, samples from all five surveyed vegetable markets were contaminated with &lambda, cyhalothrin residues at concentrations below the MRL for cabbage (MRL = 0.2 mg/kg). We conclude that &lambda, cyhalothrin residues in lettuce and cabbage from farms and markets in Parakou and Cotonou are within the MRL, and hence are relatively safe for consumption.

Background:The arbovirus vector,Aedes albopictus,originating from Asia, has recently invaded African countries, including the Republic of the Congo, where it was associated with a chikungunya outbreak. Up until now, little was known about its distribution in relation to the nativeAedes aegyptiand how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution ofAe. albopictusandAe. aegyptiin the Republic of the Congo and explored the genetic diversity of the invading species,Ae. albopictus.Methods:Immature stages ofAedeswere collected in nine locations in the Republic of the Congo in 2017 following a north-south transect and reared to adult stage. Adults were morphologically identified, counted and grouped according to species and location. Genetic diversity ofAe. albopictuswas assessed by analyzing the cytochrome oxidase I (COI) gene.Results:Ae.albopictusandAe. aegyptiwere found together across the country in all the locations investigated. The invasive species is predominant over the native species in all locations except Brazzaville, suggesting thatAe. albopictusis displacingAe. aegyptiacross Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire,Ae. albopictuswas more prevalent thanAe. aegyptiin the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity ofAe. albopictuswith only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed thatAe. albopictusfrom Congo originated from other tropical Asian countries such as China, likely as a result of increasing trade links.Conclusion:These findings are important for the implementation of vector control strategies and can serve as a foundation for further research on these vectors in the country.

Background. Malaria remains an important public health issue in Benin, with Anopheles gambiae s.l. and Anopheles funestus s.s being the predominant vectors. This study was designed to generate information on An. funestus distribution, molecular speciation, Plasmodium infection rate and insecticide susceptibility status across Benin. Methods. Mosquito samples were collected from December 2014 to January 2016 in 46 localities in Benin. These samples were mapped and An. funestus collected were speciated to the molecular level. Plasmodium infection rate was determined using a Taqman assay and susceptibility to insecticides was assessed using the WHO guidelines. The genotyping of the L119F- Gste2 mutation was also carried out. Results. An. funestus was found in 8 out of the 46 localities surveyed with a high presence in Tanongou (wet Sudanese ecological zone), Kpome, Doukonta and Pahou (sub-equatorial ecological zone). Molecular identifications revealed that only An. funestus s.s was present in southern Benin, whereas in Tanongou (northern Benin) An. funestus s.s. and An. leesoni were found in sympatry at proportions of 77.7% and 22.3% respectively. Plasmodium infection rate of An. funestus was higher in southern Benin at a range of 13 to 18% compared to 5.6% recorded in Tanongou. High DDT (8±0.5%) and permethrin (11±0.5%) resistance were observed in Doukonta, Kpome and Pahou, contrasting with relatively low resistance profiles: mortality-DDT=90±3.18% and mortality-permethrin=100% in Tanongou. Genotyping analysis revealed high frequency of the resistant 119F allele in the South (Kpome and Doukonta) compared to the North (Tanongou). Discussion and Conclusion. The high presence of An. funestus in the South compared to the North could be due to favorable environmental and climatic conditions found in both regions. A significant Plasmodium infection rate was recorded across the country. A high resistance profile was recorded in the southern Benin; this raises the need for further investigations on resistance selection factors.

Background\ud Knowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria.\ud \ud Results\ud Plasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F0 (77%) and F1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F0 and F1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F0; for F1, 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population.\ud \ud Conclusion\ud The study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.