Geographical distribution of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and genetic diversity of invading population of Ae. albopictus in the Republic of the Congo [version 1; referees: 2 approved]

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 native Aedes aegypti and how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution of Ae. albopictus and Ae. aegypti in the Republic of the Congo and explored the genetic diversity of the invading species, Ae. albopictus. Methods: Immature stages of Aedes were 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 of Ae. albopictus was assessed by analyzing the cytochrome oxidase I (COI) gene. Results: Ae. albopictus and Ae. aegypti were 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 that Ae. albopictus is displacing Ae. aegypti across Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire, Ae. albopictus was more prevalent than Ae. aegypti in the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity of Ae. albopictus with only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed that Ae. albopictus from 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

Pyrethroid resistance in the New World malaria vector Anopheles albimanus is mediated by cytochrome P450 CYP6P5

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α cypermethrin with comparable affinities (KM of 3.3 μM ± 0.4 and 3.6 μM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min− 1 ± 0.2) versus deltamethrin (2.68 min− 1 ± 0.09). Timecourse assays revealed progressive depletion of the above pyrethroids with production of four HPLCdetectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas

Molecular detection and maternal transmission of a bacterial symbiont Asaia species in field-caught Anopheles mosquitoes from Cameroon

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 F0 Asaia-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

Propiedades halocrómicas y potenciales antimicrobianos de extractos crudos de cinco plantas ornamentales

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&nbsp;pv.&nbsp;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. erecta inhibited growth of P. agglomerans without development of resistance, whereas the bacteria developed resistance against Penicillin after 18 hrs of incubation. T. erecta and A. blanchetii were able to inhibit growth of X.&nbsp;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.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&nbsp;pv.&nbsp;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 y 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. blanchetii pudieron 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

Geographical distribution of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and genetic diversity of invading population of Ae. albopictus in the Republic of the Congo

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 native Aedes aegypti and how the invasion will modify the epidemiology of arboviral diseases. Here, we assessed the current distribution of Ae. albopictus and Ae. aegypti in the Republic of the Congo and explored the genetic diversity of the invading species, Ae. albopictus. Methods: Immature stages of Aedes were 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 of Ae. albopictus was assessed by analyzing the cytochrome oxidase I (COI) gene. Results: Ae. albopictus and Ae. aegypti were 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 that Ae. albopictus is displacing Ae. aegypti across Congo. When comparing the species distributions across the two largest cities, Brazzaville and Pointe Noire, Ae. albopictus was more prevalent than Ae. aegypti in the suburbs whereas the opposite situation was reported in the city centre. Mitochondrial DNA analysis revealed very low genetic diversity of Ae. albopictus with only three haplotypes recorded across the country supporting the recent introduction of this species in the Republic of the Congo. Phylogenetic tree analysis revealed that Ae. albopictus from 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

An Experimental Hut Evaluation of PBO-Based and Pyrethroid-Only Nets against the Malaria Vector Anopheles funestus Reveals a Loss of Bed Nets Efficacy Associated with GSTe2 Metabolic Resistance

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 < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 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

The cytochrome P450 CYP325A is a major driver of pyrethroid resistance in the major malaria vector Anopheles funestus in Central Africa

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