Role of Pfs47 in the dispersal of ancestral Plasmodium falciparum malaria through adaptation to different anopheline vectors.

Plasmodium falciparum malaria originated when Plasmodium praefalciparum, a gorilla malaria parasite transmitted by African sylvan anopheline mosquitoes, adapted to humans. Pfs47, a protein on the parasite surface mediates P. falciparum evasion of the mosquito immune system by interacting with a midgut receptor and is critical for Plasmodium adaptation to different anopheline species. Genetic analysis of 4,971 Pfs47 gene sequences from different continents revealed that Asia and Papua New Guinea harbor Pfs47 haplotypes more similar to its ortholog in P. praefalciparum at sites that determine vector compatibility, suggesting that ancestral P. falciparum readily adapted to Asian vectors. Consistent with this observation, Pfs47-receptor gene sequences from African sylvan malaria vectors, such as Anopheles moucheti and An. marshallii, were found to share greater similarity with those of Asian vectors than those of vectors of the African An. gambiae complex. Furthermore, experimental infections provide direct evidence that transformed P. falciparum parasites carrying Pfs47 orthologs of P. praefalciparum or P. reichenowi were more effective at evading the immune system of the Asian malaria vector An. dirus than An. gambiae. We propose that high compatibility of ancestral P. falciparum Pfs47 with the receptors of Asian vectors facilitated the early dispersal of human malaria to the Asian continent, without having to first adapt to sub-Saharan vectors of the An. gambiae complex.

Mosquito control by abatement programmes in the United States: perspectives and lessons for countries in sub-Saharan Africa.

Africa and the United States are both large, heterogeneous geographies with a diverse range of ecologies, climates and mosquito species diversity which contribute to disease transmission and nuisance biting. In the United States, mosquito control is nationally, and regionally coordinated and in so much as the Centers for Disease Control (CDC) provides guidance, the Environmental Protection Agency (EPA) provides pesticide registration, and the states provide legal authority and oversight, the implementation is usually decentralized to the state, county, or city level. Mosquito control operations are organized, in most instances, into fully independent mosquito abatement districts, public works departments, local health departments. In some cases, municipalities engage independent private contractors to undertake mosquito control within their jurisdictions. In sub-Saharan Africa (SSA), where most vector-borne disease endemic countries lie, mosquito control is organized centrally at the national level. In this model, the disease control programmes (national malaria control programmes or national malaria elimination programmes (NMCP/NMEP)) are embedded within the central governments' ministries of health (MoHs) and drive vector control policy development and implementation. Because of the high disease burden and limited resources, the primary endpoint of mosquito control in these settings is reduction of mosquito borne diseases, primarily, malaria. In the United States, however, the endpoint is mosquito control, therefore, significant (or even greater) emphasis is laid on nuisance mosquitoes as much as disease vectors. The authors detail experiences and learnings gathered by the delegation of African vector control professionals that participated in a formal exchange programme initiated by the Pan-African Mosquito Control Association (PAMCA), the University of Notre Dame, and members of the American Mosquito Control Association (AMCA), in the United States between the year 2021 and 2022. The authors highlight the key components of mosquito control operations in the United States and compare them to mosquito control programmes in SSA countries endemic for vector-borne diseases, deriving important lessons that could be useful for vector control in SSA.

Malaria transmission pattern across the Sahelian, humid savanna, highland and forest eco-epidemiological settings in Cameroon.

BACKGROUND: Malaria remains a major public health concern in Cameroon. Understanding vector distribution and malaria transmission dynamics is of paramount importance for evaluating the performance of control strategies. This study assesses patterns of malaria transmission in four eco-epidemiological settings in Cameroon. METHODS: Adult mosquitoes were collected using Human Landing Catches (HLC) once every 4 months from August 2019 to November 2021 in Kaélé, Tibati, Santchou and Bertoua. Mosquitoes were sorted by genus and Anopheles gambiae sensu lato (s.l.) species complex were identified using PCR. The presence of Plasmodium falciparum circumsporozoite protein (CSP) was measured by ELISA; the entomological inoculation rates (EIR) was estimated in each locality. RESULTS: A total of 23,536 mosquitoes were collected. Anopheles gambiae and/or Anopheles coluzzii were the main malaria vectors in all sites. Anopheles arabiensis was recorded in low frequency in Kaélé and Tibati. Other species collected included Anopheles funestus, Anopheles pharoensis and Anopheles ziemmani. High anopheline biting rates were recorded outdoor in all sites except in Kaélé. Important differences in species biting dynamics were observed between sites. The sporozoite infection rate varied from 0.36 to 4%. The daily EIR was found to vary from 0.07 in Santchou to 0.26 infected bites/man/night (ib/m/n) in Kaélé). CONCLUSION: The study suggests heterogeneous patterns of malaria transmission in different ecoepidemiological settings across the country. The findings stress the need to improve malaria vector control strategies.

Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

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%_only, (ii): Vectomax_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.

Population knowledge, attitudes and practices towards malaria prevention in the locality of Makenene, Centre-Cameroon.

BACKGROUND: To contribute to the mission of the National Malaria Control Programme (NMCP) and guide future interventions in Cameroon in general, and in Makenene in particular, this study assessed the knowledge, attitudes and practices of the population of Makenene towards the fight against malaria. METHODS: Using a semi-structured questionnaire, a descriptive cross-sectional household community survey was carried out in randomly selected households in Makenene, a locality situated between forest and savannah ecotypes. RESULTS: Out of the 413 households surveyed, all (100%) claimed to have heard of malaria with over 94% (n = 391) associating disease transmission with mosquito bites. The main mosquito control tools used in the area were mosquito nets (92.25%). The majority of participants had good knowledge (55.93%; n = 231), good practices (71.67%, n = 296) but moderate attitudes (47.94%; n = 198) towards malaria control and fight. Good knowledge and practices were recorded mostly in educated persons including public servants and students. Good attitudes were adopted mostly by public servants and students of secondary and higher levels of education. CONCLUSION: In Makenene, the population exhibits good knowledge and practices towards malaria and its control. However, despite high LLINs ownership and use, people still complain about malaria in the area. Control tools should be monitored, repaired or replaced when necessary to support the achievement of the NMCP mission.

Pyrethroid Resistance Situation across Different Eco-Epidemiological Settings in Cameroon.

Rapid emergence and spread of pyrethroid resistance in Anopheles gambiae populations is among the main factors affecting malaria vector control in Cameroon, but there is still not enough data on the exact pyrethroid resistance status across Cameroon. The present study assessed pyrethroid resistance profile in different eco-epidemiological settings in Cameroon. Susceptibility bioassay tests were performed with F0 An. gambiae females aged three to five days. Mosquito susceptibility to both permethrin and deltamethrin was assessed. Species of the An. gambiae s.l. complex were identified using molecular diagnostic tools. Target site mutations conferring resistance were detected using Taqman assays. Quantitative reverse transcription-real-time PCR (qRT-PCR) 3-plex TaqMan® assays were used for the quantification of detoxification genes implicated in pyrethroid resistance. An. gambiae, An. coluzzii and An. arabiensis were identified in the different settings. An. gambiae was dominant in Santchou, Kékem, Bélabo, Bertoua and Njombé, while An. coluzzii was abundant in Tibati and Kaélé. High frequencies of the kdr L1014F allele ranging from 43% to 100% were recorded in almost all sites. The L1014S kdr allele was detected at low frequency (4.10-10%) only in mosquito populations from Njombé and Tibati. The N1575Y mutation was recorded in Kaélé, Santchou, Tibati and Bertoua with a frequency varying from 2.10% to 11.70%. Six Cytochrome P450 genes (Cyp6p3, Cyp6m2, Cyp9k1, Cyp6p4, Cyp6z1, and Cyp4g16) were found to be overexpressed in at least one population. Analysis of cuticular hydrocarbon lipids indicated a significant increase in CHC content in mosquito populations from Kaélé and Njombé compared to Kékem, Bélabo and Bertoua populations. The study indicated high pyrethroid resistance across different ecological settings in Cameroon with different profile of resistance across the country. The present situation calls for further actions in order to mitigate the impact of insecticide resistance on vector control measures.

Entomological and Anthropological Factors Contributing to Persistent Malaria Transmission in Kenya, Ethiopia, and Cameroon.

INTRODUCTION: In order to improve our understanding of the fundamental limits of core interventions and guide efforts based on prioritization and identification of effective/novel interventions with great potentials to interrupt persistent malaria transmission in the context of high vector control coverage, the drivers of persistent disease transmission were investigated in three eco-epidemiological settings; forested areas in Cameroon, coastal area in Kenya and highland areas in Ethiopia. METHODS: Mosquitoes were sampled in three eco-epidemiological settings using different entomological sampling techniques and analysed for Plasmodium infection status and blood meal origin in blood-fed specimens. Human behavioural surveys were conducted to assess the knowledge and attitude of the population on malaria and preventive measures, their night activities, and sleeping pattern. The parasitological analysis was conducted to determine the prevalence of Plasmodium infection in the population using rapid diagnostic tests. RESULTS: Despite the diversity in the mosquito fauna, their biting behaviour was found to be closely associated to human behaviour in the three settings. People in Kenya and Ethiopia were found to be more exposed to mosquito bites during the early hours of the evening (18-21h) while it was in the early morning (4-6 am) in Cameroon. Malaria transmission was high in Cameroon compared to Kenya and Ethiopia with over 50% of the infected bites recorded outdoors. The non-users of LLINs were 2.5 to 3 times more likely to be exposed to the risk of acquiring malaria compared to LLINs users. Malaria prevalence was high (42%) in Cameroon, and more than half of the households visited had at least one individual infected with Plasmodium parasites. CONCLUSIONS: The study suggests high outdoor malaria transmission occurring in the three sites with however different determinants driving residual malaria transmission in these areas.

Influence of house characteristics on mosquito distribution and malaria transmission in the city of Yaoundé, Cameroon.

BACKGROUND: Improving house structure is known to limit contact between humans and mosquitoes and reduce malaria transmission risk. In the present study, the influence of house characteristics on mosquito distribution and malaria transmission risk was assessed in the city of Yaoundé. METHODS: The study was conducted from March 2017 to June 2018 in 32 districts of the city of Yaoundé. Mosquito collections were performed indoor in 10 to 15 houses per district using CDC light traps. A total of 467 houses, selected randomly were used. A pretested questionnaire was submitted to participants of the study to collect information on the household: the number of people per house, education level, type of walls, presence of ceilings and eaves, number of windows, usage of long-lasting insecticidal nets (LLINs), number of bedroom and number of window. Mosquitoes collected were identified morphologically. Anophelines were tested by ELISA to detect infection by Plasmodium parasites. General Estimating Equations adjusting for repeated measures in the same house fitting negative binomial analysis were used to assess the influence of house characteristics on mosquito distribution. RESULTS: A total of 168,039 mosquitoes were collected; Culex spp emerged as the predominant species (96.48%), followed by Anopheles gambiae sensu lato (s.l.) (2.49%). Out of the 1033 An. gambiae s.l. identified by PCR, 90.03% were Anopheles coluzzii and the remaining were An. gambiae sensu stricto (s.s.) (9.97%). The high number of people per household, the presence of screens on window and the possession of LLINs were all associated with fewer mosquitoes collected indoors, whilst opened eaves, the high number of windows, the presence of holes in walls and living close to breeding sites were associated with high densities of mosquitoes indoor. Out of 3557 Anophelines tested using ELISA CSP, 80 were found infected by Plasmodium falciparum parasites. The proportion of mosquitoes infected did not vary significantly according to house characteristics. CONCLUSION: The study indicated that several house characteristics such as, the presence of holes on walls, opened eaves, unscreened window and living close to breeding sites, favored mosquito presence in houses. Promoting frequent use of LLINs and house improvement measures, such as the use of screen on windows, closing eaves, cleaning the nearby environment, should be integrated in strategies to improve malaria control in the city of Yaoundé.

Correction to: Malaria prevention in the city of Yaoundé: knowledge and practices of urban dwellers.

Following publication of the original article [1], the authors flagged an error concerning Table 3.

Malaria prevention in the city of Yaoundé: knowledge and practices of urban dwellers.

BACKGROUND: Malaria prevention in Cameroon mainly relies on the use of ITNs. Although several free distribution campaigns of treated nets have been conducted across the country, bed net usage remains very low. A household survey was conducted to assess knowledge of the population and practices affecting treated net usage in the city of Yaoundé. METHODS: A community-based descriptive cross-sectional survey was conducted in January 2017 in 32 districts of the city of Yaoundé. Parents (household head, spouse or an elder representative) who consented to the study, were interviewed using a structured pre-tested questionnaire. Interviews were conducted in French or English. A questionnaire consisting of 22 questions was administered to know (i) people's knowledge and attitude on preventive measures; and, (ii) attitudes concerning the treatment of malaria and estimated amount spent for malaria prevention and treatment. RESULTS: A total of 1643 household heads were interviewed. Over 94% of people interviewed associated malaria transmission to mosquito bites. The main methods used against mosquito bites were: treated bed nets (94%; n = 1526) and insecticide spray or coils (32.2%; n = 523). The majority of people interviewed reported using bed nets mainly to prevent from mosquito bites (84.4%, n = 1257), rather than for malaria prevention (47.3%). Knowledge and attitude analysis revealed that people with university or secondary level of education have better knowledge of malaria, prevention and treatment measures compared to those with the primary level (OR = 7.03; P < 0.001). Also, wealthy households were more aware of good practices concerning malaria prevention and treatment compared to poor ones. In the majority of districts of Yaoundé, over 50% of people interviewed per district, had good knowledge of malaria and prevention measures but less than 50% applied good practices concerning malaria treatment and prevention. The amount spent annually by a household for vector control was CFAF 11,589 ± 1133 (US$21.87 ± 2.14) and CFAF 66,403 ± 4012 (US$125.29 ± 7.57) for malaria treatment. CONCLUSION: The study indicated that, despite good knowledge of malaria and prevention measures, few people apply good practices. More sensitization needs to be done to improve adherence to good practices concerning malaria prevention and treatment.

Investigation of mechanisms of bendiocarb resistance in Anopheles gambiae populations from the city of Yaoundé, Cameroon.

BACKGROUND: Resistance to the carbamate insecticide bendiocarb is emerging in Anopheles gambiae populations from the city of Yaoundé in Cameroon. However, the molecular basis of this resistance remains uncharacterized. The present study objective is to investigate mechanisms promoting resistance to bendiocarb in An. gambiae populations from Yaoundé. METHODS: The level of susceptibility of An. gambiae s.l. to bendiocarb 0.1 % was assessed from 2010 to 2013 using bioassays. Mosquitoes resistant to bendiocarb, unexposed and susceptible mosquitoes were screened for the presence of the Ace-1(R) mutation using TaqMan assays. Microarray analyses were performed to assess the pattern of genes differentially expressed between resistant, unexposed and susceptible. RESULTS: Bendiocarb resistance was more prevalent in mosquitoes originating from cultivated sites compared to those from polluted and unpolluted sites. Both An. gambiae and Anopheles coluzzii were found to display resistance to bendiocarb. No G119S mutation was detected suggesting that resistance was mainly metabolic. Microarray analysis revealed the over-expression of several cytochrome P450 s genes including cyp6z3, cyp6z1, cyp12f2, cyp6m3 and cyp6p4. Gene ontology (GO) enrichment analysis supported the detoxification role of cytochrome P450 s with several GO terms associated with P450 activity significantly enriched in resistant samples. Other detoxification genes included UDP-glucosyl transferases, glutathione-S transferases and ABC transporters. CONCLUSION: The study highlights the probable implication of metabolic mechanisms in bendiocarb resistance in An. gambiae populations from Yaoundé and stresses the need for further studies leading to functional validation of detoxification genes involved in this resistance.

Influence of the agrochemicals used for rice and vegetable cultivation on insecticide resistance in malaria vectors in southern Côte d'Ivoire.

BACKGROUND: Vector control can contribute to the development of resistance to insecticides in malaria vectors. As the swamps and wetlands used for some agricultural activities constitute productive breeding sites for many mosquito species, agricultural pest control may increase the selection pressure for insecticide resistance in mosquitoes. Understanding the use of agrochemicals by farmers is important to plan and initiate effective integrated pest and vector management interventions. METHODS: A knowledge-attitude-practice study, using questionnaires, was undertaken with 102 rice farmers in Tiassalé and 106 vegetable farmers in Dabou (South Côte d'Ivoire) in order to generate information on pesticide usage. In addition, insecticide susceptibility bioassays were conducted using adult mosquitoes obtained from larvae collected within farms, and the persistence of agricultural pesticides in the farming environment, including sediment and mosquito breeding site water, was investigated by HPLC. RESULTS: Herbicides and insecticides appeared to be the most frequently used pesticides for both crops. Amino phosphonates (mostly glyphosate) represented the most used herbicides (45 % for rice up to 89 % for vegetables). Pyrethroids appeared to be the most used insecticides (accounting for 90 % of all the insecticide use reported). Approximately 75 % of respondents had not been to school and do not understand product labels. Only about 45 % of farmers respect the recommended pesticide dosage and about 10-15 % of pesticides used for rice and vegetable, respectively, are not recommended for these crops. As per WHO criteria, the mosquito local populations from the two localities were resistant to three of the four insecticides tested, as mortalities were less than 35 % for deltamethrin, DDT and bendiocarb. Higher susceptibility was observed for malathion, although the population was considered resistant in Dabou (80 % mortality) and susceptible in Tiassalé (98 % mortality). With the exception of glyphosate, residues from each of six chemicals tested for were detected in each of the sites visited in the two localities. CONCLUSION: The study describes the use of insecticides and herbicides on crops and highlights the importance of considering agriculture practices when attempting to manage resistance in malaria vectors. Inter-sectoral collaboration between agriculture and public health is required to develop efficient integrated pest and vector management interventions.

Distribution of Plasmodium falciparum gametocytes and malaria-attributable fraction of fever episodes along an altitudinal transect in Western Cameroon.

BACKGROUND: Highland areas are hypoendemic zones of malaria and are therefore prone to epidemics, due to lack of protective immunity. So far, Cameroon has not succeeded in implementing a convenient and effective method to detect, prevent and forecast malaria epidemic in these peculiar zones. This monitoring and evaluation study aims to assess the operational feasibility of using the human malaria infectious reservoir (HMIR) and the malaria-attributable fraction of fever episodes (MAFE) as indicators, in designing a malaria epidemic early warning system (MEWS). METHODS: Longitudinal parasitological surveys were conducted in sentinel health centres installed in three localities, located along an altitudinal transect in Western Cameroon: Santchou (750 m), Dschang (1,400 m) and Djuttitsa (1,965 m). The syndromes of outpatients with malaria-like complaints were recorded and their blood samples examined. The HMIR and the MAFE were estimated and their spatial-temporal variations described. RESULTS: The prevalence of asexual Plasmodium infection in outpatients decreased with increasing altitude; meanwhile the HMIR remained fairly constant, indicating that scarcity of malaria disease in highlands is likely due to absence of vectors and not parasites. In lowland, children carried the heaviest malaria burden in the form of febrile episodes, and asexual parasites decreased with age, after an initial peak in the 0-5 year's age group; however, they were similar for all age groups in highland. The HMIR did not show any variation with age in the plain; but some discrepancies were observed in the highland with extreme age groups, and migration of populations between lowland and highland was suspected to be the cause. Plasmodium infection was perennial in the lowland and seasonal uphill, with malaria disease occurring here mostly during the short dry season. The MAFE was high and did not change with altitude. CONCLUSION: It is obvious that a malaria outbreak will cause the sudden rise of HMIR and MAFE in highland, prior to the malaria season; the discrepancy with lowland would then help detecting an incipient malaria epidemic. It is recommended that in designing the MEWS, the National Malaria Control Programme should include these parameters and put special emphasis on: altitude, age groups and seasons.

Rapid evolution of pyrethroid resistance prevalence in Anopheles gambiae populations from the cities of Douala and Yaoundé (Cameroon).

BACKGROUND: The adaptation of malaria vectors to urban areas is becoming a serious challenge for malaria control. The study presents the evolution of pyrethroid resistance in mosquito populations from the cities of Douala and Yaoundé between 2010 and 2013. METHODS: Susceptibility tests to permethrin and deltamethrin were carried out with two- to four-day old unfed Anopheles gambiae sensu lato adults raised from larvae collected from the field. Mosquitoes resistant to permethrin and deltamethrin and control were screened to detect the presence of the kdr alleles using the TaqMan assays. Mosquitoes belonging to the An. gambiae complex were subjected to PCR assays designed for species and molecular forms identifications. The genomic region containing the upstream of intron-1 of the voltage-gated sodium channel was sequenced and compared between mosquitoes originating from different breeding habitats. RESULTS: Anopheles gambiae s.l. specimens collected from the city of Douala were all Anopheles coluzzii. In Yaoundé, both An. gambiae and An. coluzzii were recorded. A rapid decrease of mosquito mortality to permethrin and deltamethrin was recorded between 2010 and 2013 in the two cities. The mortality rate varied from 80.3 to 22.3% and 94.4 to 59.7% for permethrin and deltamethrin, respectively. Both kdr alleles L1014F and L1014S were recorded. The frequency of kdr alleles increased rapidly over the study period, varying from 44 to 88.9% in Yaoundé and from 68 to 81% in Douala. The sequencing of a 1,228 bp region of intro-1 of the voltage-gated sodium channel revealed the presence of five different haplotypes. A high number of these haplotypes were recorded in An. coluzzii samples. No evidence for a recent selective sweep on intron-1 sequence within samples originating from different breeding habitat was detected using Fu's and Tajima Fs statistics. CONCLUSION: The present study supports rapid evolution of pyrethroid resistance in vector populations from the cities of Douala and Yaoundé and calls for immediate action to fight against the increasing prevalence of pyrethroid-resistant mosquitoes.

Exposure to disinfectants (soap or hydrogen peroxide) increases tolerance to permethrin in Anopheles gambiae populations from the city of Yaoundé, Cameroon.

BACKGROUND: The rapid expansion of insecticide resistance is limiting the efficiency of malaria vector control interventions. However, current knowledge of factors inducing pyrethroid resistance remains incomplete. In the present study, the role of selection at the larval stage by disinfectants, such as soap and hydrogen peroxide (H2O2), on adult mosquito resistance to permethrin was investigated. METHODS: Field Anopheles gambiae sensu lato larvae, were exposed to variable concentrations of soap and H2O2. Larvae surviving to acute toxicity assays after 24 hours were reared to the adult stage and exposed to permethrin. The susceptibility level of adults was compared to the untreated control group. The effect of soap or hydrogen peroxide selection on the length of larval development and emergence rate was assessed. RESULT: Larval bioassays analysis showed a more acute effect of hydrogen peroxide on mosquito larvae compared to soap. The regression lines describing the dose mortality profile showed higher mean and variance to hydrogen peroxide than to soap. The duration of larval development (<5 days) and adults emergence rates (1 to 77%) were shorter and lower compare to control. Anopheles gambiae s.l. larvae surviving to selection with either soap or hydrogen peroxide or both, produced adults who were up to eight-times more resistant to permethrin than mosquitoes from the untreated control group. CONCLUSION: The present study shows that selective pressure exerted by non-insecticidal compounds such as soap and hydrogen peroxide affect An. gambiae s.l. tolerance to pyrethroids. This requires further studies with regard to the adaptation of An. gambiae s.l. to polluted habitats across sub-Saharan Africa cities.

Durability of PBO nets (Olyset Plus®), 12 months after their distribution in Bertoua, Cameroon.

Background: The rapid spread of pyrethroid resistance has led to a change in strategy, going from pyrethroid-based nets to PBO + pyrethroid-treated nets. Although these new nets may significantly improve the control of pyrethroid-resistant mosquitoes, their durability in the field remain not yet well documented. This study investigates the durability and washing resistance of Olyset-Plus nets in the city centre and rural areas of Bertoua, Cameroon. In each site, a semi-structured questionnaire was administered to at least 190 households with an Olyset-Plus net. Factors such as net use, physical integrity and bioefficacy were recorded. Bioassays were conducted on the collected nets to assess their bioefficacy and resistance to washing. They were tested against wild Anopheles gambiae sensus lato (s.l.). Unused nets and the Kisumu strain were used as controls. Washing and cone testing of the nets was carried out according to standard WHO protocols. Results: A high rate of net use by children was recorded in the urban area (89.1% (106/119)) compared to the rural area (39.7% (118/297)). The majority of Olyset-Plus nets inspected 82.2% (162/197) in the rural area and 88% (206/234) in the urban centre were in good condition (Hole Index<64). Only 5.6% and 6.8% of nets were badly torn in rural and urban sites respectively. Nets were washed more regularly in the urban centre. 88.1% of urban dwellers reported having washed their nets at least once compared to only 62% of rural dwellers. Bioefficacy tests with nets indicated a mortality rate ranging from 66% for unwashed nets to 86.7% for nets washed at least once. Bioefficacy varied significantly in the city of Bertoua according to net washing frequency, soaking time, soap type and drying location, whereas in the rural village, only washing(washed or unwashed) and soaking status (soaked or unsoaked) significantly influenced the bioefficacy of Olyset-Plus nets. Conclusions: This study revealed different handling practices of bed nets in rural and urban settings which could significantly affect Olyset-Plus nets bio-efficacy and durability. Routine monitoring and sensitization of communities to best practices concerning bed nets usage and handling during mass distribution might enhance the net durability in the community.

Physical integrity and bioefficacy of used long-lasting insecticidal nets in Makenene, Centre Region of Cameroon.

Long-lasting insecticide nets (LLINs) are the recommended tools against mosquito-borne diseases. However, their physical integrity and bioefficacy in the field could be affected by several factors. This study evaluated the physical integrity and bioefficacy of nets used in Makenene since 2016. Cross-sectional field surveys were carried out after 6 y. A questionnaire was first administered to the heads of households, and then the physical integrity of the LLINs was determined by calculating the proportional hole index (pHI). WHO cone bioassays were conducted to determine the bioefficacy of LLINs currently being used against wild strains of Anopheles gambiae s.l., Culex pipiens s.l., and laboratory-reared pyrethroid-susceptible strain of Anopheles coluzzii (Ngousso). Of the 167 LLINs examined in households, 39.5% were fairly good, 26.4% were acceptable, and 34.1% were damaged. The most torn faces of the nets were the sides used for entering and exiting. None of the 30 LLINs used for WHO cone bioassays was still effective against An. gambiae s.l. and Cx. pipiens s.l. while up to 85.7% of these LLINs were at least effective against the susceptible strain after 24 h, with a significant difference observed when comparing the mortality rates between wild and laboratory-susceptible strain of Anopheles (P-value < 0.01). Anopheles gambiae s.l. were all (100%) identified as An. gambiae s.s. by PCR. The LLINs distributed in Makenene since the 2016 campaign are only effective on susceptible strain and should be replaced for a better control of residual malaria transmission and the nuisance by Culex mosquitoes in the locality.

First detection of kdr L1014F allele in Anopheles ziemanni and Anopheles pharoensis in Cameroon and distribution of the allele in members of the Anopheles gambiae complex.

BACKGROUND: Knockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented. METHODS: The present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing. RESULTS: An. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote "RS" state in An. arabiensis and An. gambiae in Yangah and Santchou. CONCLUSIONS: The present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission.

Physiological correlates of ecological divergence along an urbanization gradient: differential tolerance to ammonia among molecular forms of the malaria mosquito Anopheles gambiae.

BACKGROUND: Limitations in the ability of organisms to tolerate environmental stressors affect their fundamental ecological niche and constrain their distribution to specific habitats. Evolution of tolerance, therefore, can engender ecological niche dynamics. Forest populations of the afro-tropical malaria mosquito Anopheles gambiae have been shown to adapt to historically unsuitable larval habitats polluted with decaying organic matter that are found in densely populated urban agglomerates of Cameroon. This process has resulted in niche expansion from rural to urban environments that is associated with cryptic speciation and ecological divergence of two evolutionarily significant units within this taxon, the molecular forms M and S, among which reproductive isolation is significant but still incomplete. Habitat segregation between the two forms results in a mosaic distribution of clinally parapatric patches, with the M form predominating in the centre of urban agglomerates and the S form in the surrounding rural localities. We hypothesized that development of tolerance to nitrogenous pollutants derived from the decomposition of organic matter, among which ammonia is the most toxic to aquatic organisms, may affect this pattern of distribution and process of niche expansion by the M form. RESULTS: Acute toxicity bioassays indicated that populations of the two molecular forms occurring at the extremes of an urbanization gradient in Yaounde, the capital of Cameroon, differed in their response to ammonia. The regression lines best describing the dose-mortality profile differed in the scale of the explanatory variable (ammonia concentration log-transformed for the S form and linear for the M form), and in slope (steeper for the S form and shallower for the M form). These features reflected differences in the frequency distribution of individual tolerance thresholds in the two populations as assessed by probit analysis, with the M form exhibiting a greater mean and variance compared to the S form. CONCLUSIONS: In agreement with expectations based on the pattern of habitat partitioning and exposure to ammonia in larval habitats in Yaounde, the M form showed greater tolerance to ammonia compared to the S form. This trait may be part of the physiological machinery allowing forest populations of the M form to colonize polluted larval habitats, which is at the heart of its niche expansion in densely populated human settlements in Cameroon.

Diverse novel Wolbachia bacteria strains and genera-specific co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon.

Background: The endosymbiotic bacterium Wolbachia infects numerous species of insects and Wolbachia transinfection of Aedes mosquito species is now being used for biocontrol programs as Wolbachia strains can both inhibit arboviruses and invade wild mosquito populations. The discovery of novel, resident Wolbachia strains in mosquito species warrants further investigation as potential candidate strains for biocontrol strategies. Methods: We obtained mosquito specimens from diverse Culicine mosquitoes from Cameroon including ecologically diverse locations in the Central and West Regions. Wolbachia prevalence rates were assessed in addition to the environmentally acquired bacterial species Asaia in major Culicine genera. PCR-based methods were also used with phylogenetic analysis to confirm identities of host mosquito species and Wolbachia strains were classified using multi-locus sequence typing (MLST). Results: We report high Wolbachia prevalence rates for Culicine species, including in a large cohort of Aedes africanus collected from west Cameroon in which 100% of mono-specific pools were infected. Furthermore, co-infections with Asaia bacteria were observed across multiple genera, demonstrating that these two bacteria can co-exist in wild mosquito populations.  Wolbachia strain MLST and phylogenetic analysis provided evidence for diverse Wolbachia strains in 13 different mosquito species across seven different genera.  Full or partial MLST profiles were generated from resident Wolbachia strains in six Culex species ( quinquefasciatus, watti, cinerus, nigripalpus, perexiguus and rima), two Aedes species (africanus and denderensis) and in Mansonia uniformis, Catageiomyia argenteopunctata, Lutzia tigripes, Eretmapodites chrysogaster and Uranotaenia bilineata. Conclusions: Our study provides further evidence that Wolbachia is widespread within wild mosquito populations of diverse Culicine species and provides further candidate strains that could be investigated as future options for Wolbachia-based biocontrol to inhibit arbovirus transmission.