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Background: Gaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. Here, we provide evidence demonstrating that two highly selected mutations in CYP6P4a and CYP6P4b are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa., Results: Continent-wide polymorphism survey revealed escalated signatures of directional selection of both genes between 2014 and 2021. In vitro insecticide metabolism assays with recombinant enzymes from both genes showed that mutant alleles under selection exhibit higher metabolic efficiency than their wild-type counterparts. Using the GAL4-UAS expression system, transgenic Drosophila flies overexpressing mutant alleles exhibited increased resistance to pyrethroids. These findings were consistent with in silico predictions which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we designed two DNA-based assays for the detection of CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce killing effects of pyrethroid bed nets., Conclusions: Overall, this study demonstrated that CYP6P4a and CYP6P4b contribute to pyrethroid resistance in An. funestus and provided two additional insecticide resistance molecular diagnostic tools that would contribute to monitoring and better management of resistance., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Insecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides., Results: Our analysis revealed limited population structure within Northern and Central regions (F ST <0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns (F ST >0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations (F ST >0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region (F ST =0.29). Evidence of selective sweeps was supported by negative Tajima's D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere., Conclusion: Our study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon., Competing Interests: Declarations. Ethics approval and consent to participate: This study underwent review and approval by the National Ethics Committee for Health Research (CNERSH) of Cameroon, with the identification number 2021/07/1372/CE/CNERSH/SP. Chiefs from the villages where mosquitoes were collected, as well as the house owners, gave their consent before collection was done. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Problem Addressed: Crimean Congo hemorrhagic fever (CCHF) is a tick-borne disease with high fatality rates and an expansive geographic distribution, yet disease prevalence data in Cameroon is lacking., Objective: This study aimed to determine CCHF virus (CCHFV) seroprevalence and tick distribution among cattle herders and febrile patients in West and Centre Cameroon., Methods and Approach: Two cross-sectional serological studies of human and cattle were conducted from October to December 2021 and from June to July 2022, which included the collection of ticks. Enzyme-linked immunosorbent assay (ELISA) were used to detect anti-CCHFV antibodies, while a knowledge, attitudes, and practice (KAP) survey assessed tick and tickborne disease related knowledge and behaviors among herders. Tick identification used morphological keys., Results: The KAP survey showed adequate tick knowledge (94.5 %) among herders but poor understanding of disease transmission, with favorable attitudes towards tick control (24.7 %) but inadequate implementation. Rhipicephalus annulatus (64.1 %) predominated among the 1,296 ticks collected during each rainy season. Among cattle, 27.4 % were seropositive, and seropositivity was associated with specific villages, cattle age (>4 years), and female sex. Herders had a 17.8 % seroprevalence, while febrile patients had 8.3 %, with higher rates in those >20 years old for both groups. Self-reported tick removal by herders after contact and grazing may increase CCHFV exposure., Conclusions: This study confirms CCHFV circulation in rural West Cameroon and unexpected exposure risk in Yaounde, highlighting the need for active entomological surveillance and preventive measures in transhumance and cattle market activities. Establishing an occupation-based surveillance system can help identify CCHFV hotspots to prevent outbreaks., 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 Elsevier B.V. All rights reserved.)

The widespread prevalence of Asaia in mosquitoes makes it a potential candidate for paratrangenic control in Anopheles . To better understand whether this bacterium could be used for malaria control, we quantified Asaia in An. gambiae s.l populations in malaria endemic regions examining co-infection with Plasmodium falciparum . Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during both the dry and wet seasons. DNA was extracted from whole individual mosquitoes, and real time-qPCR amplification of the 16S ribosomal RNA was used to quantify Asaia in both An. gambiae and An. coluzzii samples. We also detected and quantified P. falciparum infection in the same mosquitoes. The density of Asaia was successfully quantified in a total of 864 field mosquitoes, comprising of 439 An. gambiae from Bankeng and 424 An. coluzii collected from Gounougou. Interestingly, a higher prevalence of Asaia in An. gambiae (88.3%) compared to An. coluzzii (80.9%) was observed. Moreover, the density of Asaia in both species was significantly affected by seasonal changes in the two localities. Furthermore, a significant difference between the infection densities of Asaia and the Plasmodium infection status in the two species was recorded. However, no correlation was observed between the number of Asaia and P. falciparum infections. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii . Nevertheless, further studies incorporating experimental infections are required to better investigate the correlation between Anopheles mosquitoes, Asaia, and Plasmodium .IMPORTANCEThe symbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but further analysis of its biology and genetics across Africa is necessary. In this study, we investigated and quantified the influence of Asaia in naturally infected An. gambiae s.l . populations with the malaria parasite Plasmodium falciparum . Genomic DNA was extracted from whole individual mosquitoes collected from two localities, and Asaia was quantified using real-time qPCR by amplification of the 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in the same mosquitoes and established the correlation between Asaia and Plasmodium coinfection. This study provides evidence that naturally occurring Asaia infection is not correlated with P. falciparum development within An. gambiae and An. coluzzii mosquitoes.

Metabolic mechanisms conferring pyrethroid resistance in malaria vectors are jeopardizing the effectiveness of insecticide-based interventions, and identification of their markers is a key requirement for robust resistance management. Here, using a field-lab-field approach, we demonstrated that a single mutation G454A in the P450 CYP9K1 is driving pyrethroid resistance in the major malaria vector Anopheles funestus in East and Central Africa. Drastic reduction in CYP9K1 diversity was observed in Ugandan samples collected in 2014, with selection of a predominant haplotype (G454A mutation at 90%), which was completely absent in the other African regions. However, six years later (2020) the Ugandan 454A-CYP9K1 haplotype was found predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). Comparative in vitro heterologous expression and metabolism assays revealed that the mutant 454A-CYP9K1 (R) allele significantly metabolises more type II pyrethroid (deltamethrin) compared with the wild G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing 454A-CYP9K1 (R) allele exhibited significantly higher type I and II pyrethroids resistance compared to flies expressing the wild G454-CYP9K1 (S) allele. Furthermore, laboratory testing and field experimental hut trials in Cameroon demonstrated that mosquitoes harbouring the resistant 454A-CYP9K1 allele significantly survived to pyrethroids exposure (Odds ratio = 567, p < 0.0001). This study highlights the rapid spread of pyrethroid resistant CYP9K1 allele, under directional selection in East and Central Africa, contributing to reduced bed net efficacy. The newly designed DNA-based assay here will add to the toolbox of resistance monitoring and improving its management strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Novel vector control tools against African trypanosomiases require a deep understanding of the factors driving tsetse vector fitness or population resilience in their ecosystems. Following evidence of microbiota-mediated host fitness or traits shaping, including insecticide resistance in arthropod populations, we undertook a comparative study of the microbiota in wild-caught tsetse flies during vector control with deltamethrin-impregnated traps called Tiny Targets. The bacterial microbiome composition of tsetse flies collected before and after 6, 12, and 18 months of vector control were characterized using high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene and compared. Overall, 48 bacterial genera and five phyla were identified. The primary symbiont Wigglesworthia dominated almost all the samples with an overall relative abundance of 71.76%. A significant increase was observed in microbiome diversities over the vector control with new taxa identified. Interestingly, few genera, like Curvibacter for instance, displayed a regularly increasing abundance, from 0.57% to 0.65%, 4.73%, and 8.57% after 6, 12, and 18 months of tsetse control, respectively. This study provided preliminary for further investigation into the role and mechanism of action of microbiota in tsetse fly fitness under selective pressure like insecticides.IMPORTANCEThe interest in vector control in the fight against African trypanosomiases has been reinforced in recent years, with the development of small insecticide-impregnated screens, known as "Tiny Targets". As some tsetse biotopes are difficult to access for their installation, other tools are under consideration that involve using bacteria harbored by the tsetse vector to block the development of trypanosomes or impair the tsetse's fitness in its natural environment. Several bacterial symbionts were previously described as important for tsetse fly development, and some like Burkholderia and Citrobacter also found in tsetse flies were found associated with insecticide tolerance in other arthropods. In this research, we found the bacterial genera, Curvibacter and Acinetobacter, increased in abundance in tsetse flies during vector control. These bacteria deserve further attention to determine if they can interfere with insecticides used to control tsetse fly populations., Competing Interests: The authors declare no conflict of interest.

Background: Insecticides are a crucial component of vector control. However, resistance constitute a threat on their efficacy and the gains obtained over the years through malaria vector control. In Gabon, little data on phenotypic insecticide resistance in Anopheles vectors are published, compromising the rational implementation of resistance management strategies. We assessed the susceptibility to pyrethroids, carbamates and organophosphates of Anopheles gambiae sensu lato (s.l.) and discuss the mechanisms involved in the pyrethroid resistance-phenotype., Methods: A. gambiae s.l. larvae were collected from breeding sites in Lambaréné. Emerging adults were used in WHO tube assays at an insecticide concentration that defines resistance (diagnostic concentration). Subsequently, deltamethrin and permethrin were used at 5x and 10x diagnostic concentrations and after preexposure with the cytochrome p450 (and glutathione S-transferase) inhibitor piperonyl butoxide (PBO). A subset of mosquitoes was typed by molecular methods and screened using Taqman assays for mutations conferring target site resistance at the Voltage-gated sodium channel 1014 (Vgsc-1014) locus and the acetylcholinesterase (Ace-1) gene., Results: All mosquitoes were A. gambiae sensu stricto (s.s.) and resistant to permethrin, deltamethrin and alphacypermethrin (mortality less than 98%). However, mosquitoes were susceptible to malathion but resistant to bendiocarb. The level of resistance was high for permethrin and at least moderate for deltamethrin. Pre-exposure to PBO significantly increased the mortality of resistant mosquitoes (P < 0.0001). They became fully susceptible to deltamethrin and permethrin-induced mortality increased 4-fold. The G119S Ace-1 resistance allele, which confers resistance to both organophosphates and carbamates, was not present. All sampled mosquitoes were either homozygous for the Vgsc-L1014F or heterozygous for Vgsc-L1014F/L1014S, a marker for resistance to pyrethroids and organochlorides., Conclusion: These findings demonstrate a role of cytochrome P450 monooxygenases in the pyrethroid-resistance of A. gambiae s.s. from Lambaréné. Combining PBO with pyrethroids, as done in second generation bednets, may be used to revert resistance. In addition, malathion could also be used in combination with pyrethroids-based methods for resistance management., (© 2024. The Author(s).)

Improved understanding of mosquito-plant feeding interactions can reveal insights into the ecological dynamics of pathogen transmission. In wild malaria vectors Anopheles gambiae s.l. and An. funestus group surveyed in selected dryland ecosystems of Kenya, we found a low level of plant feeding (2.8%) using biochemical cold anthrone test but uncovered 14-fold (41%) higher rate via DNA barcoding targeting the chloroplast rbcL gene. Plasmodium falciparum positivity was associated with either reduced or increased total sugar levels and varied by mosquito species. Gut analysis revealed the mosquitoes to frequently feed on acacia plants (~ 89%) (mainly Vachellia tortilis) in the family Fabaceae. Chemical analysis revealed 1-octen-3-ol (29.9%) as the dominant mosquito attractant, and the sugars glucose, sucrose, fructose, talose and inositol enriched in the vegetative parts, of acacia plants. Nutritional analysis of An. longipalpis C with high plant feeding rates detected fewer sugars (glucose, talose, fructose) compared to acacia plants. These results demonstrate (i) the sensitivity of DNA barcoding to detect plant feeding in malaria vectors, (ii) Plasmodium infection status affects energetic reserves of wild anopheline vectors and (iii) nutrient content and olfactory cues likely represent potent correlates of acacia preferred as a host plant by diverse malaria vectors. The results have relevance in the development of odor-bait control strategies including attractive targeted sugar-baits., (© 2024. The Author(s).)

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mugenzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Elevated resistance to pyrethroids in major malaria vectors has led to the introduction of novel insecticides including neonicotinoids. There is a fear that efficacy of these new insecticides could be impacted by cross-resistance mechanisms from metabolic resistance to pyrethroids. In this study, after evaluating the resistance to deltamethrin, clothianidin and mixture of clothianidin + deltamethrin in the lab using CDC bottle assays, the efficacy of the new IRS formulation Fludora® Fusion was tested in comparison to clothianidin and deltamethrin applied alone using experimental hut trials against wild free-flying pyrethroid-resistant Anopheles funestus from Elende and field An. gambiae collected from Nkolondom reared in the lab and released in the huts. Additionally, cone tests on the treated walls were performed each month for a period of twelve months to evaluate the residual efficacy of the sprayed products. Furthermore, the L1014F-kdr target-site mutation and the L119F-GSTe2 mediated metabolic resistance to pyrethroids were genotyped on a subset of mosquitoes from the EHT to assess the potential cross-resistance. All Anopheles species tested were fully susceptible to clothianidin and clothianidin + deltamethrin mixture in CDC bottle assay while resistance was noted to deltamethrin. Accordingly, Fludora® Fusion (62.83% vs 42.42%) and clothianidin (64.42% vs 42.42%) induced significantly higher mortality rates in EHT than deltamethrin (42.42%) against free flying An. funestus from Elende in month 1 (M1) and no significant difference in mortality was observed between the first (M1) and sixth (M6) months of the evaluation (P > 0.05). However, lower mortality rates were recorded against An. gambiae s.s from Nkolondom (mortality rates 50%, 45.56% and 26.68%). In-situ cone test on the wall showed a high residual efficacy of Fludora® Fusion and clothianidin on the susceptible strain KISUMU (> 12 months) and moderately on the highly pyrethroid-resistant An. gambiae strain from Nkolondom (6 months). Interestingly, no association was observed between the L119F-GSTe2 mutation and the ability of mosquitoes to survive exposure to Fludora® Fusion, whereas a trend was observed with the L1014F-kdr mutation. This study highlights that Fludora® Fusion, through its clothianidin component, has good potential of controlling pyrethroid-resistant mosquitoes with prolonged residual efficacy. This could be therefore an appropriate tool for vector control in several malaria endemic regions., (© 2024. The Author(s).)

The versatility of cytochrome P450 reductase ( CPR ) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR ( AgCPR ), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR ( AfCPR ) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR , e.g., the coordinates of the second FAD stacking residue AfCPR -Y 456 differ from that of AgCPR -His 456 . While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR , the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b , capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning., (© 2024 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

UDP-glycosyltransferases (UGTs) enzymes are pivotal in insecticide resistance by transforming hydrophobic substrates into more hydrophilic forms for efficient cell elimination. This study provides the first comprehensive investigation of Anopheles funestus UGT genes, their evolution, and their association with pyrethroid resistance. We employed a genome-wide association study using pooled sequencing (GWAS-PoolSeq) and transcriptomics on pyrethroid-resistant An. funestus, along with deep-targeted sequencing of UGTs in 80 mosquitoes Africa-wide. UGT310B2 was consistently overexpressed Africa-wide and significant gene-wise F st differentiation was observed between resistant and susceptible populations: UGT301C2 and UGT302A3 in Malawi, and UGT306C2 in Uganda. Additionally, nonsynonymous mutations in UGT genes were identified. Gene-wise Tajima's D density curves provide insights into population structures within populations across these countries, supporting previous observations. These findings have important implications for current An. funestus control strategies facilitating the prediction of cross-resistance to other UGT-metabolised polar insecticides, thereby guiding more effective and targeted insecticide resistance management efforts., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S -transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S -transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p  < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p  < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p  < 0.0114), CYP4G16 (FC = 2.02, p  < 0.0005), and CYP4G17 (FC = 1.86, p  < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace -1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon., Competing Interests: The authors declare no financial and ethical conflicts of interest., (© 2024 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Background: Chronic exposure of mosquito larvae to pesticide residues and cross-resistance mechanisms are major drivers of tolerance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid prequalified for Indoor Residual Spraying (IRS)., Methods: Using standard bioassays, we tested if reduced susceptibility to clothianidin can affect the efficacy of SumiShield® 50WG, one of four new IRS formulations containing clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp sampled from urban, suburban and agricultural areas of Yaoundé, Cameroon., Results: We found that in this geographic area, the level of susceptibility to the active ingredient predicted the efficacy of SumiShield 50WG. This formulation was very potent against populations that reached 100% mortality within 72 h of exposure to a discriminating concentration of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield 50WG in An. gambiae adults collected from a farm where the spraying of the two neonicotinoids acetamiprid and imidacloprid for crop protection is likely driving resistance to clothianidin., Conclusions: Despite the relatively small geographic extend of the study, the findings suggest that cross-resistance may impact the efficacy of some new IRS formulations and that alternative compounds could be prioritized in areas where neonicotinoid resistance is emerging., (© 2024. The Author(s).)

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5-15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A 16 I 51 R 59 N 108 I 164 . The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V 431 A 436 G 437 K 540 A 581 A 613 alleles (20.8%). Similarly, the Pmdhfr N 50 K 55 L 57 R 58 S 59 S 114 F 168 I 170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps - S 436 A 437 occured at 37.2% frequency. The combined quadruple N 50 K 55 L 57 R 58 S 59 S 114 F 168 I 170 &#95; S 436 G 437 K 540 A 581 A 613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems., Competing Interests: The authors declare no conflict of interest.

Background: Significant progress has been made towards African sleeping sickness elimination in the last decade. Indeed, the World Health Organization (WHO) global goal of eliminating the chronic form of the disease as a public health problem was achieved in 2020 (i.e., < 2,000 new cases per year). Vector control has played an important role in achieving this goal. In this study, we evaluated the impact of the insecticide impregnated Tiny Targets on tsetse fly densities and their infection rates with Trypanosoma spp in the Campo sleeping sickness focus of South Cameroon., Methods: The study site was divided into two areas: (i) the south-west experimental area, which included vector control, and (ii) the eastern part as the non-intervention area. After compiling the baseline entomological data (tsetse densities and trypanosome infection rates), around 2000 Tiny Targets were deployed in the South-West area and replaced every six months for two years. Post-intervention surveys were conducted every six months to determine tsetse densities and levels of trypanosome infections with PCR-based methods., Results: Following the intervention, tsetse mean catches decreased by 61% after six months, and up to 73% after twelve months (pre-intervention: 2.48 flies/trap/day, 95%CI [1.92-3.14]; 12-months post-intervention: 0.66 tsetse/trap/day, 95%CI [0.42-0.94]). This decrease was not sustained after 18 months, and the mean catch doubled compared to that after 12 months. After 24 months, the mean catches still increased by 17% (18 months: 1.45 tsetse/trap/day, 95%CI [1.07-1.90] and 24 months: 1.71 tsetse/trap/day, 95%CI [1.27-2.24]). In the non-intervention area, a variation in tsetse catches was observed during the two years, with a general increase from 2.43 [0.73-5.77] to 3.64 [1.47-7.70] tsetse/trap/day. In addition, trypanosome infection rates dropped by 75% in both areas (P-value < 0.001) from 21.20% to 5.06% and from 13.14% to 3.45% in intervention and control areas respectively., Conclusion: Tiny targets have proven useful in reducing tsetse population densities and trypanosome infection rates, providing evidence for the integration of this tool in current strategies towards trypanosomiasis elimination in Campo. The non-sustained decrease of tsetse densities after one year may indicate reinvasions from neighbouring breeding sites or that the intervention area was not large enough. Our results show the need to scale up by accessing difficult breeding sites and extend the tiny targets to the whole transborder focus., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Melachio Tanekou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies., (© The Author(s) 2023. 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 impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos' propensity to transmit Plasmodium spp., Methods: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes., Results: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1-5.2), p = 0.03; IRR = 2.5, 95% CI (1.2-5.8), p = 0.01 respectively)., Conclusion: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance., (© 2023. The Author(s).)

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control., (© 2023. Springer Nature Limited.)

Background: Larviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids., Methodology: The susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%&#95;only, (ii): Vectomax&#95;only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain "Ngousso". Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding., Results: An increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate., Conclusion: The profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Inc.)

Background: The increasing reports of resistance to pyrethroid insecticides associated with reduced efficacy of pyrethroid-only interventions highlight the urgency of introducing new non-pyrethroid-only control tools. Here, we investigated the performance of piperonyl-butoxide (PBO)-pyrethroid [Permanet 3.0 (P3.0)] and dual active ingredients (AI) nets [Interceptor G2 (IG2): containing pyrethroids and chlorfenapyr and Royal Guard (RG): containing pyrethroids and pyriproxyfen] compared to pyrethroid-only net Royal Sentry (RS) against pyrethroid-resistant malaria vectors in Cameroon., Methods: The efficacy of these tools was firstly evaluated on Anopheles gambiae s.l. and Anopheles funestus s.l. from Gounougou, Mibellon, Mangoum, Nkolondom, and Elende using cone/tunnel assays. In addition, experimental hut trials (EHT) were performed to evaluate the performance of unwashed and 20 times washed nets in semi-field conditions. Furthermore, pyrethroid-resistant markers were genotyped in dead vs alive, blood-fed vs unfed mosquitoes after exposure to the nets to evaluate the impact of these markers on net performance. The XLSTAT software was used to calculate the various entomological outcomes and the Chi-square test was used to compare the efficacy of various nets. The odds ratio and Fisher exact test were then used to establish the statistical significance of any association between insecticide resistance markers and bed net efficacy., Results: Interceptor G2 was the most effective net against wild pyrethroid-resistant An. funestus followed by Permanet 3.0. In EHT, this net induced up to 87.8% mortality [95% confidence interval (CI): 83.5-92.1%) and 55.6% (95% CI: 48.5-62.7%) after 20 washes whilst unwashed pyrethroid-only net (Royal Sentry) killed just 18.2% (95% CI: 13.4-22.9%) of host-seeking An. funestus. The unwashed Permanet 3.0 killed up to 53.8% (95% CI: 44.3-63.4%) of field-resistant mosquitoes and 47.2% (95% CI: 37.7-56.7%) when washed 20 times, and the Royal Guard 13.2% (95% CI: 9.0-17.3%) for unwashed net and 8.5% (95% CI: 5.7-11.4%) for the 20 washed net. Interceptor G2, Permanet 3.0, and Royal Guard provided better personal protection (blood-feeding inhibition 66.2%, 77.8%, and 92.8%, respectively) compared to pyrethroid-only net Royal Sentry (8.4%). Interestingly, a negative association was found between kdrw and the chlorfenapyr-based net Interceptor G2 (χ 2  = 138; P < 0.0001) with homozygote-resistant mosquitoes predominantly found in the dead ones., Conclusions: The high mortality recorded with Interceptor G2 against pyrethroid-resistant malaria vectors in this study provides first semi-field evidence of high efficacy against these major malaria vectors in Cameroon encouraging the implementation of this novel net for malaria control in the country. However, the performance of this net should be established in other locations and on other major malaria vectors before implementation at a large scale., (© 2023. National Institute of Parasitic Diseases.)

Background: The double burden of malaria and helminthiasis in children poses an obvious public health challenge, particularly in terms of anemia morbidity. While both diseases frequently geographically overlap, most studies focus on mono-infection and general prevalence surveys without molecular analysis. The current study investigated the epidemiological determinants of malaria, schistosomiasis, and geohelminthiasis transmission among children in the North Region of Cameroon., Methodology: School and pre-school children aged 3-15 year-of-age were enrolled from three communities in March 2021 using a community cross-sectional design. Capillary-blood samples were obtained, and each was examined for malaria parasites using rapid-diagnostic-test (RDT), microscopy, and PCR while hemoglobin level was measured using a hemoglobinometer. Stool samples were analyzed for Schistosoma mansoni, S. guineensis, and soil-transmitted-helminthiasis (STH) infections using the Kato Katz method, and urine samples were assessed for the presence of S. haematobium eggs (including hybrids) using the standard urine filtration technique., Result: A malaria prevalence of 56% (277/495) was recorded by PCR as opposed to 31.5% (156/495) by microscopy and 37.8% (186/495) by RDT. Similarly, schistosomiasis was observed at prevalence levels of up to 13.3% (66/495) overall [S. haematobium (8.7%); S. mansoni (3.8%); mixed Sh/Sm (0.6%); mixed Sh/Sm/Sg (0.2%). Both infections were higher in males and the 3-9 year-of-age groups. A high frequency of PCR reported P. falciparum mono-infection of 81.9% (227/277) and mixed P. falciparum/P. malariae infection of 17.3% (48/277) was observed. Malaria-helminths co-infections were observed at 13.1% (65/495) with marked variation between P. falciparum/S. haematobium (50.8%, 33/65); P. falciparum/S. mansoni (16.9%, 11/65) and P. falciparum/Ascaris (9.2%, 6/65) (χ2 = 17.5, p = 0.00003). Anemia prevalence was 32.9% (163/495), categorically associated with P. falciparum (45.8%, 104/227), Pf/Sh (11.5%, 26/227), and Pf/Sm (3.9%, 9/227) polyparasitism., Conclusion: Polyparasitism with malaria and helminth infections is common in school-aged children despite periodic long-lasting insecticide-treated nets (LLINs) distribution and regular school-based praziquantel (for schistosomiasis) and albendazole (for STH) campaigns. Co-existence of Plasmodium parasites and helminths infections notably Schistosoma species among children may concurrently lead to an increase in Plasmodium infection with an enhanced risk of anemia, highlighting the necessity of an integrated approach for disease control interventions., Competing Interests: he authors have declared that no competing interests exist., (Copyright: © 2023 Nkemngo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although Culex species are considered to be equally affected by control measures targeting malaria vectors, there is still not enough evidence of the impact of interventions such as larviciding on the distribution of these mosquito species. The present study assessed the impact of a larviciding trial targeting malaria vectors on Culex mosquito species in the city of Yaoundé, Cameroon. A cluster randomized trial comparing 13 treated clusters and 13 untreated clusters was implemented. Data were collected at baseline and during the larviciding intervention, from March 2017 to November 2020. The microbial larvicide VectoMax G was applied once every 2 weeks in the intervention areas. Adult mosquitoes were collected using CDC light traps in both intervention and non-intervention areas and compared between arms. Globally, larviciding intervention was associated with 69% reduction in aquatic habitats with Culex larvae and 36.65% reduction of adult Culex densities in houses. Adult Culex densities were reduced both indoors (35.26%) and outdoors (42.37%). No change in the composition of Culex species was recorded. The study suggests a high impact of larviciding on Culex mosquito species distribution. The impact of the intervention can be improved if typical Culex breeding habitats including pit latrines are targeted., Competing Interests: 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., (© 2023 The Authors.)

Background: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon., Results: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450., Conclusions: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination., (© 2023. The Author(s).)

Evaluating the susceptibility of malaria vectors to the new WHO-recommended products is a key step before large-scale deployment. We mapped the susceptibility profile of Anopheles funestus to neonicotinoids across Africa and established the diagnostic doses of acetamiprid and imidacloprid with acetone + MERO as solvent. Indoor resting An. funestus were collected in 2021 in Cameroon, Malawi, Ghana and Uganda. Susceptibility to clothianidin, imidacloprid and acetamiprid was evaluated using CDC bottle assays and offsprings of the field-caught adults. The L119F-GSTe2 marker was genotyped to assess the potential cross-resistance between clothianidin and this DDT/pyrethroid-resistant marker. Mosquitoes were susceptible to the three neonicotinoids diluted in acetone + MERO, whereas low mortality was noticed with ethanol or acetone alone. The doses of 6 µg/mL and 4 µg/mL were established as diagnostic concentrations of imidacloprid and acetamiprid, respectively, with acetone + MERO. Pre-exposure to synergists significantly restored the susceptibility to clothianidin. A positive correlation was observed between L119F-GSTe2 mutation and clothianidin resistance with the homozygote resistant mosquitoes being more able to survive than heterozygote or susceptible. This study revealed that An. funestus populations across Africa are susceptible to neonicotinoids, and as such, this insecticide class could be effectively implemented to control this species using IRS. However, potential cross-resistance conferred by GSTe2 calls for regular resistance monitoring in the field.

Introduction: Despite a high fatality rate in humans, little is known about the occurrence of Crimean-Congo hemorrhagic fever virus (CCHFV) in Cameroon. Hence, this pioneer study was started with the aim of determining the prevalence of CCHFV in domestic ruminants and its potential vector ticks in Cameroon., Methods: A cross-sectional study was carried out in two livestock markets of Yaoundé to collect blood and ticks from cattle, sheep, and goats. CCHFV-specific antibodies were detected in the plasma using a commercial ELISA assay and confirmed using a modified seroneutralization test. Ticks were screened for the presence of orthonairoviruses by amplification of a fragment of the L segment using RT-PCR. Phylogeny was used to infer the genetic evolution of the virus., Results: Overall, 756 plasma samples were collected from 441 cattle, 168 goats, and 147 sheep. The seroprevalence of CCHFV was 61.77% for all animals, with the highest rate found in cattle (433/441, 98.18%) followed by sheep (23/147, 15.65%), and goats (11/168, 6.55%), ( p -value < 0.0001). The highest seroprevalence rate was found in cattle from the Far North region (100%). Overall, 1500 ticks of the Rhipicephalus (773/1500, 51.53%), Amblyomma (341/1500, 22.73%), and Hyalomma (386/1500, 25.73%) genera were screened. CCHFV was identified in one Hyalomma truncatum pool collected from cattle. Phylogenetic analysis of the L segment classified this CCHFV strain within the African genotype III., Conclusion: These seroprevalence results call for additional epidemiological studies on CCHFV, especially among at-risk human and animal populations in high-risk areas of the country., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Simo Tchetgna, Yousseu, Cosset, de Freitas, Kamgang, McCall, Ndip, Legros and Wondji.)

Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC<7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a&#95;susceptible (e.g Odds ratio = 20.8, P<0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P<0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mugenzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microbiome composition has been associated with insecticide resistance in malaria vectors. However, the contribution of major symbionts to the increasingly reported resistance escalation remains unclear. This study explores the possible association of a specific endosymbiont, Asaia spp., with elevated levels of pyrethroid resistance driven by cytochrome P450s enzymes and voltage-gated sodium channel mutations in Anopheles funestus and Anopheles gambiae. Molecular assays were used to detect the symbiont and resistance markers ( CYP6P9a/b , 6.5 kb , L1014F , and N1575Y ). Overall, genotyping of key mutations revealed an association with the resistance phenotype. The prevalence of Asaia spp. in the FUMOZ&#95;X&#95;FANG strain was associated with the resistance phenotype at a 5X dose of deltamethrin (OR = 25.7; p = 0.002). Mosquitoes with the resistant allele for the markers tested were significantly more infected with Asaia compared to those possessing the susceptible allele. Furthermore, the abundance correlated with the resistance phenotype at 1X concentration of deltamethrin ( p = 0.02, Mann-Whitney test). However, for the MANGOUM&#95;X&#95;KISUMU strain, findings rather revealed an association between Asaia load and the susceptible phenotype ( p = 0.04, Mann-Whitney test), demonstrating a negative link between the symbiont and permethrin resistance. These bacteria should be further investigated to establish its interactions with other resistance mechanisms and cross-resistance with other insecticide classes.

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance., (© 2023. The Author(s).)

Background: Malaria control efforts are highly skewed towards Plasmodium falciparum while overlooking other Plasmodium species such as P. malariae. A better understanding of the role of Plasmodium species other than P. falciparum is needed to strengthen malaria elimination initiatives. The aim of the present study was to elucidate the contribution of P. malariae to malaria transmission in Cameroon., Methods: The study was conducted in the Ngatti Health District, a forest-savannah transition area in the Adamawa Region, Cameroon. A total of 497 individuals aged from 1 to 85 years were diagnosed with malaria in November 2020 using a rapid diagnostic test (RDT) and microscopy. Adult mosquitoes were collected between September 2019 and March 2020 by indoor aspiration and identified morphologically and molecularly. The infection status of Plasmodium spp. was also determined by quantitative PCR, and dried blood spots were collected from 156 participants with the aim to detect different Plasmodium species by nested PCR., Results: The overall Plasmodium prevalence was 50.3%, 51.8% and 64.7%, as detected by microscopy, the RDT and PCR, respectively. Based on the PCR results, P. falciparum was the most prevalent species (43%); followed by co-infections P. falciparum/P. malariae (17%), P. falciparum/P. ovale (1.3%), P. falciparum/P. ovale/P. malariae (1.3%); and then by P. malariae mono-infection (2.5%). The same trend was observed using microscopy, with 35% of participants infected with P. falciparum, 11% co-infected with P. falciparum/P. malariae and 4% infected with P. malariae. The prevalence and parasite density of malaria infection varied significantly with age group (P < 0.05), with the highest prevalence rate observed in children aged 6-10 years (P = 0.0001) while the density of Plasmodium infection increased significantly in children aged < 5 years compared to the other age groups (P = 10 -3 ). Among the 757 Anopheles mosquitoes collected, 737 (97.35%) were An. funestus sensu stricto, 15 (1.9%) were An. gambiae and 5 (0.6%) were An. hancocki. The Plasmodium species recorded at the head/thorax level were P. falciparum and P. malariae, with a sporozoite infection rate of 8.4%; the highest sporozoite infection rate was recorded at Mibellon village (13.6%)., Conclusion: The results of this study reveal the significant contribution of P. malariae, in addition to P. falciparum, to the high malaria transmission rate in this region. These findings highlight the need to deploy initiatives to also tackle this Plasmodium species to eliminate malaria in the region., (© 2023. The Author(s).)

Assessing the genetic diversity of metabolic resistance genes, such as cytochrome P450s, helps to understand the dynamics and evolution of resistance in the field. Here, we analyzed the polymorphisms of CYP6M2 and CYP6P4, associated with pyrethroid resistance in Anopheles coluzzii and Anopheles gambiae , to detect potential resistance markers. Field-caught resistant mosquitos and susceptible lab strains were crossed, and F4 was exposed to permethrin for 15 min and 90 min to discriminate highly susceptible (HS) and highly resistant (HR) mosquitos, respectively. Significant permethrin mortality reduction was observed after pre-exposure to PBO, suggesting the gene involvement of P450s. qPCR analysis revealed significant overexpression of CYP6M2 ( FC = 19.57 [95% CI 13.96-25.18] for An. coluzzii ; 10.16 [7.86-12.46] for An. gambiae ) and CYP6P4 ( FC = 6.73 [6.15-7.30] An. coluzzii ; 23.62 [26.48-20.76] An. gambiae ). Full-gene and ≈1 kb upstream were sequenced. For CYP6M2, the upstream region shows low diversity in HR and HS (overall Hd = 0.49, π = 0.018), whereas the full-gene shows allelic-variation but without evidence of ongoing selection. CYP6P4 upstream region showed a lower diversity in HR (Hd = 0.48) than HS (Hd = 0.86) of An. gambiae . These results highlighted that CYP6P4 -associated resistance is potentially driven by modification in upstream region. However, further work is needed to determine the real causative variants that will help design rapid detection tools.

Introduction: Population factors such as urbanization, socio-economic, and environmental factors are driving forces for emerging/re-emerging zoonotic diseases in Cameroon. To inform preparedness and prioritization efforts, this study mapped out epidemiological data (including prevalence) of zoonotic diseases occurring in Cameroon between 2000 and 2022 by demographic factors., Methods: Following the PRISMA guidelines, a protocol was registered in the PROSPERO database (CRD42022333059). Independent reviewers searched the PubMed, Embase, CINAHL, Cochrane, and Scopus databases on May 30, 2022 for relevant articles; duplicates were removed, and the titles, abstracts, and full texts were screened to identify eligible articles., Results: Out of 4142 articles identified, 64 eligible articles were retrieved in the database search and an additional 12 from the cited literature ( N  = 76). Thirty-five unique zoonoses (viral, bacterial, and parasitic) were indexed, including Cameroon priority zoonoses: anthrax, bovine tuberculosis, Ebola and Marburg virus disease, highly pathogenic avian influenza, and rabies. The number of studies varied by region, ranging from 12 in the Far North to 32 in the Centre Region. The most reported were as follows: brucellosis (random-effects pooled estimate proportion (effect size), ES 0.05%, 95% confidence interval (CI) 0.03-0.07; n  = 6), dengue (ES 0.13%, 95% CI 0.06-0.22; n  = 12), avian and swine influenza virus (ES 0.10%, 95% CI 0.04-0.20; n  = 8), and toxoplasmosis (ES 0.49%, 95% CI 0.35-0.63; n  = 11), although I 2 values were greater than 75%, thus there was high inter-study heterogeneity ( P < 0.01)., Conclusions: This understanding of the distribution of emerging and re-emerging zoonotic threats in Cameroon is vital to effective preventive and resource prioritization measures., (© 2022 The Author(s).)

Monitoring the trend of insecticide resistance and understanding associated genetic mechanisms is important for designing efficient malaria vector control strategies. This study was conducted to provide temporal data on insecticide resistance status and mechanisms in the major malaria vector Anopheles gambiae s.l. from Ebolowa, Southern Cameroon. Methods: Larvae of An. gambiae s.l. were collected from typical breeding sites throughout the city and reared to adulthood. Emerging adults were morphologically identified and WHO tube assays were performed to determine their susceptibility to carbamate, organophosphate and pyrethroid insecticides at diagnostic doses. When resistance was observed, its intensity was determined by performing WHO tube tests using 5 and 10 times the concentration of the diagnostic dose. Metabolic resistance mechanisms were investigated using insecticide-synergist assays. Sibling species of the An. gambiae complex were identified using SINE-PCR protocol. TaqMan assay was used to genotype the L1014F and L1014S kdr mutations, and the N1575Y mutation, an amplifier of the resistance conferred by the L1014F mutation. Results: Anopheles coluzzii was by far the dominant (99%) member of the An. gambiae s.l. complex in Ebolowa. The species was fully susceptible to carbamates and organophosphates, but resistant to all pyrethroid insecticides tested. Resistance was of moderate intensity for deltamethrin (mortality: 37%, 70% and 99% for 1×, 5× and 10× insecticide concentration, respectively) but rather of high intensity for permethrin (5% for 1×; 62% for 5× and 75% for 10×) and for alphacypermethrin (4.4% for 1×; 57% for 5× and 80% for 10×). Pre-exposure to the synergist PBO resulted in a full recovery of the susceptibility to delthametrin, but this was not observed for the other two pyrethroids tested. L1014S (kdr-East) and the N1575Y mutations were absent, whereas the L1014F (kdr-West) mutation was present at a high frequency (75%), showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9−7.4]; p < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6−5.4]; p = 0.0002). Conclusion: The increased resistance of An. gambiae s.l. to pyrethroid insecticides as observed in Ebolowa poses a threat to the efficacy of LLINs used to protect populations from the bites of Anopheles mosquitoes that transmit malaria parasites. The present study further highlights the urgent need to implement resistance management strategies in order to maintain the effectiveness of insecticide-based vector control interventions and prevent a rebound in malaria-related mortality.

Purpose: Myxozoans are economically important group of metazoan parasites, which can cause diseases in a large variety of commercially important fishes. Increased knowledge on molecular features has shown that traditional descriptive characters may be misleading. Combination of both descriptive and molecular features is therefore necessary for an integrated taxonomic assessment., Methods: Cyprinid Labeo batesii, sampled in the Makombè River at Nkondjock in Cameroon were examined for myxosporeans. Identification of parasite species was based on morphological and molecular sequence analyses of myxospores. Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian inference (BI) methods., Results: The scales of L. batesii were infected by Myxobolus nkondjockei sp. nov Their mature myxospores are ovoid in frontal view and lenticular in lateral view, with two rounded ends. These myxospores measured 10.3 (10-10.9) μm length and 8.0 (7.3-8.5) μm width. Myxospores have two ovoid and equal sizes polar capsules. They measured 4.5 (4.0-5.0) µm in length and 2.4 (2-2.9) µm in width. Polar tubules were coiled in 4-5 turns perpendicular to the longitudinal axis of the polar capsules. Phylogenetic analysis of the 18S rDNA sequence show clustering of M. nkondjockei sp. n. close to an undetermined species Myxobolus sp. reported infecting gill lamellas of Labeo rohita from India., Conclusion: The morphological, molecular and phylogenetic data provided for M. nkondjockei sp. n. are solid basis for further identification of this myxozoan of which pathogenicity probably plays an economic role at culturing the hosts., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Background: Urban malaria is becoming a major public health concern in major cities in Cameroon. To improve malaria vector control, a pilot larviciding trial was conducted to assess its impact on mosquito density and malaria transmission intensity in Yaoundé. The present study investigated perceptions and practices of communities on malaria control during the larviciding trial implemented in Yaoundé., Methods: Quantitative and qualitative data were collected in non-intervention and intervention areas. Quantitative data were collected during three cross-sectional surveys using a structured pre-tested questionnaire while qualitative data were obtained through interviews. A total of 26 in-depth interviews and eight focus group discussions with community members were performed. A binary logistic regression model was used to assess the perception of the community on larviciding impact on some malaria or bed nets use indicators., Results: People living in intervention areas were 2.64 times more likely to know the mode of malaria transmission (95% CI: 1.82-3.84; p<0.001) and 1.3 time more likely to know mosquito breeding habitats (95% CI: 1.06-1.56; p = 0.009) compared to those living in non-intervention areas. In intervention areas, interviewee opinions on larviciding were generally good i.e. most interviewees reported having noticed a reduction in mosquito nuisance and malaria cases following larviciding implementation; whereas in non-intervention areas no report of reduction of mosquito nuisance was recorded. LLINs were regularly used by the population despite the implementation of larviciding treatments. There was high interest in larviciding program and demand for continuation, even if this needs the community involvement., Conclusion: The larviciding program in the city of Yaoundé did not negatively affected community members' behaviour and practices concerning the use of treated nets. The study indicated the acceptance of larviciding program by the population. This positive environment could favour the implementation of future antilarval control activities in the city of Yaoundé., Competing Interests: The authors have declared that no competing interests exist.

Background: Community presence of loiasis must be determined before mass drug administration programmes for lymphatic filariasis and onchocerciasis can be implemented. However, taking human blood samples for loiasis surveillance is invasive and operationally challenging. A xenosurveillance approach based on the molecular screening of mosquitoes and their excreta/feces (E/F) for Loa loa DNA may provide a non-invasive method for detecting the community presence of loiasis., Methods: We collected 770 wild mosquitoes during a pilot study in a known loiasis transmission area in Mbalmayo, Cameroon. Of these, 376 were preserved immediately while 394 were kept in pools to collect 36-hour E/F samples before processing. Carcasses and E/F were screened for L. loa DNA. To demonstrate this method's potential for integrated disease surveillance, the samples were further tested for Wuchereria bancrofti, Mansonella perstans, and Plasmodium falciparum., Results: Despite limited sample numbers, L. loa DNA was detected in eight immediately-stored mosquitoes (2.13%; 95% CI 1.08 to 4.14), one carcass stored after providing E/F (0.25%; 95% CI 0.04 to 1.42), and three E/F samples (estimated prevalence 0.77%; 95% CI 0.15 to 2.23%). M. perstans and P. falciparum DNA were also detected in carcasses and E/F samples, while W. bancrofti DNA was detected in E/F. None of the carcasses positive for filarial worm DNA came from pools that provided a positive E/F sample, supporting the theory that, in incompetent vectors, ingested parasites undergo a rapid, complete expulsion in E/F., Conclusions: Mosquito xenosurveillance may provide a useful tool for the surveillance of loiasis alongside other parasitic diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations., Methods: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F 0 and F 1 populations using PCR and qPCR methods., Results: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s., Conclusions: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries., (© 2022. The Author(s).)

(1) Background: Schistosomiasis remains a public health issue in Cameroon. Snail control using Niclosamide can prevent schistosome transmission. It is safe to determine lethal concentrations for the population. This study aimed at assessing the toxicity of Niclosamide on different developmental stages of snail populations; (2) Methods: Snails were collected, identified, and reared in the laboratory. Egg masses and adult snails were exposed to Niclosamide, at increasing concentrations (0.06, 0.125, 0.25, 0.5, 1 mg/L for egg embryos and 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2 mg/L for adults). After 24 h exposure, egg masses and snails were removed from Niclosamide solutions, washed with source water and observed; (3) Results: Snail susceptibility was species and population dependent. For egg embryos, Biomphalaria pfeifferi was the most susceptible (LC 50 : 0.1; LC 95 : 6.3 mg/L) and Bulinus truncatus the least susceptible (LC 50 : 4.035; LC 95 : 228.118 mg/L). However, for adults, B. truncatus was the most susceptible (mortality rate: 100%). The LC 50 and LC 95 for Bi. camerunensis eggs were 0.171 mg/L and 1.102 mg/L, respectively, and were higher than those obtained for adults (0.0357 mg/L and 0.9634 mg/L); (4) Conclusion: These findings will guide the design of vector control strategies targeting these snail species in Cameroon.

Constant assessment of insecticide resistance levels is mandatory to implement adequate malaria control tools, but little information is available on the annual dynamics of resistance. We, therefore, monitored variations in resistance in Anopheles gambiae s.l. over four seasons during 2 years in two localities of Yaoundé: urban Etoa-Meki and peri-urban Nkolondom. Mosquitoes were collected seasonally at larval stage and reared to adults for insecticide susceptibility tests and molecular analysis of resistance mechanisms. Anopheles coluzzii was found in Etoa-Meki and An. gambiae in Nkolondom. Low mortalities to pyrethroids were observed (permethrin <10%, deltamethrin <21%), and resistance extended to 5× and 10× diagnostic doses, revealing a marked increase compared to previous studies. A seasonal variation in resistance was observed with the highest levels within dry seasons in Etoa-Meki and rainy seasons in Nkolondom. The 1014F kdr allele shows a high frequency (0.9), associated with overexpression of metabolic genes (Cyp6M2, Cyp6P4, Cyp9K1, Cyp6Z1, and Cyp6Z2) varying significantly seasonally. This study reveals an escalation in resistance to pyrethroids in Yaoundé's malaria vectors with seasonal variations. An adequate choice of the implementation period of punctual vector control actions according to the resistance profile will help to potentiate the desired effect and thus improve its efficiency., (© 2022 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Sleeping sickness is still prevalent in Campo, southern Cameroon, despite the efforts of World Health Organization and the National Control Programme in screening and treating cases. Reducing disease incidence still further may need the control of tsetse vectors. We update entomological and parasitological parameters necessary to guide tsetse control in Campo. Tsetse flies were trapped, their apparent densities were evaluated as the number of flies captured per trap per day and mapped using GIS tools. Polymerase chain reaction based methods were used to identify their trypanosome infection rates. Glossina palpalis palpalis was the dominant vector species representing 93.42% and 92.85% of flies captured respectively during the heavy and light dry seasons. This species presented high densities, that is, 3.87, 95% CI [3.84-3.91], and 2.51, 95% CI [2.49-2.53] flies/trap/day in the two seasons. Moreover, 16.79% (of 1054) and 20.23% (of 1132 flies) were found infected with at least 1 trypanosome species for the 2 seasons respectively, Trypanosoma congolense being the most prevalent species, and Trypanosoma. brucei gambiense identified in 4 samples. Tsetse flies are abundant in Campo and present high trypanosome infection rates. The detection of tsetse infected with human trypanosomes near the newly created palm grove show workers' exposition. Tsetse densities maps built will guide vector control with 'Tiny Targets'., (© 2022 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Little is known about the impact of ticks on livestock and humans in Cameroon. This study aimed to determine the prevalence, seasonal variation, and genetic diversity of hard ticks in the country. Ticks were collected during a cross-sectional survey on domestic livestock in two markets of Yaoundé in 2019 and 2020 and identified using morphological keys, 16S ribosomal DNA, (16S rDNA), and the cytochrome c oxidase subunit 1 (Cox1) genes. The infestation rates were 39.18%, 11.53%, and 2.74% in cattle, sheep, and goats respectively. Three genera of ticks were identified, Rhipicephalus, Amblyomma, and Hyalomma comprising eleven tick species. The main species were Rhipicephalus decoloratus (30.25%), R. microplus (24.43%), and Amblyomma variegatum (12.96%). Rhipicephalus spp. (81.31%) and Amblyomma variegatum (51.54%) were abundant during the rainy season, while Hyalomma spp. (83.86%) during the dry season (p-value <0.00001). Cox1 and 16S rDNA analysis showed a high level of genetic diversity among tick species with sequences close to those observed across Africa. Phylogenetic analysis revealed that our R. microplus belong to clade A and we identified R. sanguineus s.l. as R. linnea. This study shows a high tick infestation rate in cattle, while low in small ruminants with an extensive diversity of tick species, including several known vectors of important tick-borne diseases., (© 2022 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Understanding the environmental factors affecting the microbiota in malaria vectors may help in the development of novel vector control interventions, similar to paratransgenesis. This study evaluated seasonal and geographical variations in the microbial community of the two major malaria vectors. Adult Anopheles mosquitoes were collected across two different eco-geographical settings in Cameroon, during the dry and wet seasons. DNA was extracted from the whole individual mosquitoes from each group and processed for microbial analysis using Illumina Miseq sequencing of the V3-V4 region of the 16S rRNA gene. Data analysis was performed using QIIME2 and R software programs. A total of 1985 mosquitoes were collected and among them, 120 were selected randomly corresponding to 30 mosquitoes per season and locality. Overall, 97 bacterial taxa were detected across all mosquito samples, with 86 of these shared between dry and wet seasons in both localities and species. There were significant differences in bacterial composition between both seasons, with a clear separation observed between the dry and wet seasons (PERMANOVA comparisons of beta diversity, Pseudo-F = 10.45; q-value = 0.01). This study highlights the influence of seasonal variation on microbial communities and this variation's impact on mosquito biology and vectorial capacity should be further investigated., (© 2022 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Background: Dengue (DENV), chikungunya (CHIKV) and Zika virus (ZIKV), are mosquito-borne viruses of medical importance in most tropical and subtropical regions. Vector control, primarily through insecticides, remains the primary method to prevent their transmission. Here, we evaluated insecticide resistance profiles and identified important underlying resistance mechanisms in populations of Aedes aegypti and Ae. albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations., Methods: Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation, G1. Ae. aegypti and Ae. albopictus adults from G1 were tested following World Health Organization (WHO) recommendations and Ae. aegypti G0 adults screened with real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Piperonyl butoxide (PBO) assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance. Statistical analyses were performed using Chi-square test and generalized linear models., Results: Loss of susceptibility was observed to all insecticides tested. Mortality rates from tests with 0.25% permethrin varied from 24.27 to 85.89% in Ae. aegypti and from 17.35% to 68.08% in Ae. albopictus. Mortality rates for 0.03% deltamethrin were between 23.30% and 88.20% in Ae. aegypti and between 69.47 and 84.11% in Ae. albopictus. We found a moderate level of resistance against bendiocarb, with mortality rates ranging from 69.31% to 90.26% in Ae. aegypti and from 86.75 to 98.95% in Ae. albopictus. With PBO pre-exposure, we found partial or fully restored susceptibility to pyrethroids and bendiocarb. The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae. aegypti populations from Douala sites resistant to permethrin and deltamethrin. Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae. albopictus samples. F1534C and V1016I mutations were detected in A. aegypti mosquitoes and for the first time V410L was reported in Cameroon., Conclusions: This study revealed that Ae. aegypti and Ae. albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated. These findings could guide insecticide use to control arbovirus vectors in Cameroon., (© 2022. The Author(s).)

Background: Rift Valley fever (RVF) is a mosquito-borne zoonosis endemic in Africa. With little known of the burden or epidemiology of RVF virus (RVFV) in Cameroon, this study aimed to determine the seroprevalence of RVFV in domestic ruminants of various origins in two markets of Yaoundé, Cameroon., Methodology/principal Findings: The origin of animals randomly sampled at two livestock markets in Yaoundé were recorded and plasma samples collected for competitive and capture Enzyme-linked Immunosorbent Assay (ELISA) to determine the prevalence of Immunoglobulins G (IgG) and Immunoglobulins M (IgM) antibodies. Following ELISA IgM results, a real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect RVFV RNA. In June-August 2019, February-March 2020, and March-April 2021, 756 plasma samples were collected from 441 cattle, 168 goats, and 147 sheep. RVFV IgG seroprevalence was 25.7% for all animals, 42.2% in cattle, 2.7% in sheep, and 2.4% in goats. However, IgM seroprevalence was low, at 0.9% in all animals, 1.1% in cattle, 1.4% in sheep, and 0% in goats. The seroprevalence rates varied according to the animal's origin with the highest rate (52.6%) in cattle from Sudan. In Cameroon, IgG and IgM rates respectively were 45.1% and 2.8% in the North, 44.8% and 0% in the Adamawa, 38.6% and 1.7% in the Far-North. All IgM positive samples were from Cameroon. In cattle, 2/5 IgM positive samples were also IgG positive, but both IgM positive samples in sheep were IgG negative. Three (42.9%) IgM positive samples were positive for viral RVFV RNA using qRT-PCR but given the high ct values, no amplicon was obtained., Conclusion/significance: These findings confirm the circulation of RVFV in livestock in Cameroon with prevalence rates varying by location. Despite low IgM seroprevalence rates, RVF outbreaks can occur without being noticed. Further epidemiological studies are needed to have a broad understanding of RVFV transmission in Cameroon., Competing Interests: The authors have declared that no competing interests exist.

Plasmodium falciparum and Plasmodium malariae infections are prevalent in malaria-endemic countries. However, very little is known about their interactions especially the effect of P. malariae on P. falciparum genetic diversity. This study aimed to assess P. falciparum genetic diversity in P. falciparum and mixed infection P. falciparum/P. malariae isolates among the asymptomatic populations in Southern Benin. Two hundred and fifty blood samples (125 of P. falciparum and 125 P. falciparum/P. malariae isolates) were analysed by a nested PCR amplification of msp1 and msp2 genes. The R033 allelic family was the most represented for the msp1 gene in mono and mixed infection isolates (99.2% vs 86.4%), while the K1 family had the lowest frequency (38.3% vs 20.4%). However, with the msp2 gene, the two allelic families displayed similar frequencies in P. falciparum isolates while the 3D7 allelic family was more represented in P. falciparum/P. malariae isolates (88.7%). Polyclonal infections were also lower (62.9%) in P. falciparum/P. malariae isolates (p < 0.05). Overall, 96 individual alleles were identified (47 for msp1 and 49 for msp2) in P. falciparum isolates while a total of 50 individual alleles were identified (23 for msp1 and 27 for msp2) in P. falciparum/P. malariae isolates. The Multiplicity of Infection (MOI) was lower in P. falciparum/P. malariae isolates (p < 0.05). This study revealed a lower genetic diversity of P. falciparum in P. falciparum/P. malariae isolates using msp1 and msp2 genes among the asymptomatic population in Southern Benin., (Copyright © 2022 Elsevier B.V. All rights reserved.)