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Abstract Background A fast development of urban agriculture has recently taken place in many areas in the Republic of Benin. This study aims to assess the rapid expansion of urban agriculture especially, its contribution to the emergence of insecticide resistance in populations of Anopheles gambiae. Methods The protocol was based on the collection of sociological data by interviewing vegetable farmers regarding various agricultural practices and the types of pesticides used. Bioassay tests were performed to assess the susceptibility of malaria vectors to various agricultural insecticides and biochemical analysis were done to characterize molecular status of population of An. gambiae. Results This research showed that: (1) The rapid development of urban agriculture is related to unemployment observed in cities, rural exodus and the search for a balanced diet by urban populations; (2) Urban agriculture increases the farmers' household income and their living standard; (3) At a molecular level, PCR revealed the presence of three sub-species of An. gambiae (An. gambiae s.s., Anopheles melas and Anopheles arabiensis) and two molecular forms (M and S). The kdr west mutation recorded in samples from the three sites and more specifically on the M forms seems to be one of the major resistance mechanisms found in An. gambiae from agricultural areas. Insecticide susceptibility tests conducted during this research revealed a clear pattern of resistance to permethrin (76% mortality rate at Parakou; 23.5% at Porto-Novo and 17% at Cotonou). Conclusion This study confirmed an increase activity of the vegetable farming in urban areas of Benin. This has led to the use of insecticide in an improper manner to control vegetable pests, thus exerting a huge selection pressure on mosquito larval population, which resulted to the emergence of insecticide resistance in malaria vectors.

Abstract Background Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes. Results All mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations. Conclusion Multiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes.

Abstract Background The emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for further studies on the mechanisms of insecticide resistance and the various factors selecting resistant populations of mosquitoes. This research targeted the analysis of breeding sites and the oviposition behaviour of susceptible and resistant populations of Anopheles in localities of spilled petroleum products. The aim was to establish the possible contribution of oil spillage in the selection of pyrethroid resistance in malaria vectors. Methods Anopheles breeding sites were identified and the insecticide susceptibility of the Anopheles gambiae populations mapped in 15 localities of South Western Nigeria. The presence of oil particles as well as the turbidity, the dissolved oxygen and the pH of each identified breeding site was recorded. Data were cross-analysed to correlate the habitat types and the insecticide susceptibility status of emerging mosquitoes. The second phase of this study was basically a laboratory model to provide more information on the implication of the spillage of petroleum on the selection of pyrethroid resistance in An. gambiae. Results Moderate levels of resistance following exposure to permethrin-impregnated papers were recorded with the majority of An. gambiae samples collected in the South Western Nigeria. Data from this study established a link between the constituency of the breeding sites and the resistance status of the emerging Anopheles. Conclusion This study has revealed the segregational occupation of breeding habitats by pyrethroid resistant and susceptible strains of An. gambiae in south-western Nigeria. Compiled results from field and laboratory research point out clear relationships between oil spillage and pyrethroid resistance in malaria vectors. The identification of this factor of resistance could serve as strong information in the management of insecticide resistance in some West African settings.

Abstract Background The emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for developing new insecticidal molecules or, improving the efficacy of existing insecticides or abandoning those to which resistance has emerged. The use of petroleum products (PP) against mosquito larvae had an immense success during early programmes of malaria control, but these compounds were abandoned and replaced in the 1950s by synthetic insecticides probably because of the high performances given by these new products. In the current context of vector resistance, it is important to elucidate the empirical use of PP by quantifying their efficiencies on resistant strains of Anopheles. Methods Larvae of Anopheles Ladji a local resistant strain were exposed to increasing concentrations of various PP (kerosene, petrol and engine oils) for 24 hours and the lethal activities recorded. The highest concentration (HiC) having no lethal activity (also referred as the NOEL or no effect level) and the lowest concentration (LoC100) yielding 100% mortality were rated for each PP on the Ladji strain. Prior to laboratory analysis, KAP studies were conducted in three traditional communities were insecticide resistance is clearly established to confirm the use of PP against mosquitoes. Results Laboratory analysis of petrol, kerosene and engine oils, clearly established their lethal activities on resistant strains of Anopheles larvae. Contrary to existing references, this research revealed that exposed larvae of Anopheles were mostly killed by direct contact toxicity and not by suffocation as indicated in some earlier reports. Conclusion This research could serve as scientific basis to backup the empirical utilisation of PP on mosquito larvae and to envisage possibilities of using PP in some traditional settings where Anopheles have developed resistance to currently used insecticides.

Abstract Background The role of agricultural practices in the selection of insecticide resistance in malaria vectors has so far been hypothesized without clear evidence. Many mosquito species, Anopheles gambiae in particular, lay their eggs in breeding sites located around agricultural settings. There is a probability that, as a result of farming activities, insecticide residues may be found in soil and water, where they exercise a selection pressure on the larval stage of various populations of mosquitoes. To confirm this hypothesis, a study was conducted in the Republic of Benin to assess the environmental hazards which can be generated from massive use of pesticides in agricultural settings. Methods Lacking an HPLC machine for direct quantification of insecticide residues in samples, this investigation was performed using indirect bioassays focussed on the study of factors inhibiting the normal growth of mosquito larvae in breeding sites. The speed of development was monitored as well as the yield of rearing An. gambiae larvae in breeding sites reconstituted with water and soil samples collected in agricultural areas known to be under pesticide pressure. Two strains of An. gambiae were used in this indirect bioassay: the pyrethroid-susceptible Kisumu strain and the resistant Ladji strain. The key approach in this methodology is based on comparison of the growth of larvae in test and in control breeding sites, the test samples having been collected from two vegetable farms. Results Results obtained clearly show the presence of inhibiting factors on test samples. A normal growth of larvae was observed in control samples. In breeding sites simulated by using a few grams of soil samples from the two vegetable farms under constant insecticide treatments (test samples), a poor hatching rate of Anopheles eggs coupled with a retarded growth of larvae and a low yield of adult mosquitoes from hatched eggs, was noticed. Conclusion Toxic factors inhibiting the hatching of anopheles eggs and the growth of larvae are probably pesticide residues from agricultural practices. Samples used during this indirect assay have been stored in the laboratory and will be analysed with HPLC techniques to confirm hypothesis of this study and to identify the various end products found in soil and water samples from agricultural settings under pesticide pressure.

Background: Anopheles s.l. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness. This study investigated the larval metal exposure on larval development and reproduction of An. gambiae s.s., a laboratory susceptible strain, kisumu., Methods: Stage 2 larvae of Anopheles gambiae , Kisumu were exposed to C 1 = 484 μg L -1 , C 2 = 300 μg L -1 and 0 μg L -1 (control) of copper chloride. Larval mortality, pupation time, pupation rate, gonotrophic cycle length, fecundity and fertility of larvae/adults were assessed over six generations., Results: Results revealed that larval mortality rate was significantly higher in the C 1 groups of each group (p = 0.000), but this mortality rate decreased over generations. Pupation time was extended to 13 and 14 days respectively for C 2 and C 1 groups (p = 0.000) compared to the control group. Similar results were observed for the gonotrophic cycle, which increased from 4 days at G0 to more than 6 days at generation 5 in adults of C 1 . The pupation rate in generation 4 (C 1 ) and generation 5 of the same group (p = 0.000) as well as the emergence rate in generation 4 (C 2 , p = 0.000) and generation 5 (C 1 and C 2 , p = 0.000) decreased significantly compared to the control group. The average number of eggs laid was lower in the test groups from generation 4 to generation 5 (C 1 and C 2 , p = 0.00) and egg fertility was also negatively affected by exposure of the larval stage of An. gambiae s.s. to copper., Conclusion: This study showed that copper not only exhibits larvicidal properties in Anopheles gambiae s.s. larvae, it also revealed the potential of this metal to reduce fecundity and fertility in these malaria vectors., Competing Interests: No competing interests were disclosed., (Copyright: © 2025 Koto Yérima Gounou Boukari M et al.)

Mycotoxins such as aflatoxins (AFs), fumonisins (FBs), zearalenone (ZEN), and deoxynivalenol (DON) pose a risk to public health due to their carcinogenic potency (AFs and FBs) and anti-nutritional effects. The hazards associated with mycotoxins are accentuated where food management practices, control, and regulatory systems from farm to plate are sub-optimal. Information on the frequency of these mycotoxins in rice commercialized in markets in sub-Sahara Africa (SSA) is limited. The current study examined AF concentrations in 527 rice samples collected from 54 markets in five SSA countries. Grain quality characteristics, processing methods, and origin of samples were contrasted with toxin levels. In total, 72% of the samples had detectable AFs levels (range = 3.0 to 89.8 µg/kg). Forty-seven percent (47%) of the samples had AFs above 4 µg/kg, the European Union maximum level (ML), and were evaluated for cooccurrence with FBs, ZEN, and DON. Total AFs and ZEN cooccurred in 40% of the samples, and 30% of the positive ZEN samples had concentrations above the ML of 75 µg/kg. Total AFs did not co-occur with FBs and DON. Multivariate analysis revealed that length-to-width ratio (p < 0.0001), mixed variety for width (p = 0.04), and chalkiness (p = 0.009) significantly influenced aflatoxin concentrations. Slender grains had higher AFs concentrations than bold and medium grains (p < 0.0001). Possible strategies to mitigate mycotoxin contamination in rice include improving grain quality traits and practicing proper drying and hermetic storage before and after milling. These findings provide valuable insights for both domestic and international actors in establishing and strengthening regulations and management systems to mitigate rice mycotoxin contamination., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethics approval: This study does not involve any human or animal testing. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them., (© 2024. The Author(s).)

Agricultural pesticides may play a crucial role in the selection of resistance in field populations of mosquito vectors. This study aimed to determine the susceptibility level of An. gambiae s.l. to pyrethroids and neonicotinoids in vegetable farms in northern Benin, in West Africa, and the underlying insecticide resistance mechanisms. A survey on agricultural practices was carried out on 85 market gardeners chosen randomly in Malanville and Parakou. Anopheles gambiae s.l. larvae were collected, reared to adult stages, and identified to species level. Susceptibility was tested with impregnated papers (WHO bioassays) or CDC bottles according to the insecticides. Synergists (PBO, DEM, and DEF) were used to screen resistance mechanisms. Allelic frequencies of the kdr (L1014F) , kdr (L1014S) , N1575Y , and ace-1R G119S mutations were determined in mosquitoes using Taqman PCR. Fertilizers and pesticides were the agrochemicals most used with a rate of 97.78% and 100%, respectively, in Malanville and Parakou. Anopheles coluzzii was the predominant species in Malanville, while An. gambiae was the only species found in Parakou. Bioassays revealed a high resistance of An. gambiae s.l. to pyrethroids and DDT, while a susceptibility to bendiocarb, pyrimiphos-methyl, malathion, and clothianidin was recorded. Resistance to acetamiprid was suspected in mosquitoes from both localities. A lower resistance level was observed when mosquitoes were pre-treated with synergists, then exposed to insecticides. The kdr L1014F mutation was observed in both locations at moderate frequencies (0.50 in Malanville and 0.55 in Parakou). The allelic frequencies of N1575Y and G119S were low in both study sites. This study confirmed the resistance of An. gambiae s.l. to insecticides used in agriculture and public health. It reveals a susceptibility of vectors to bendiocarb, pyrimiphos-methyl, malathion, and clothianidin, thus indicating that these insecticides can be used as an alternative in Benin to control malaria vectors.

Background: To overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile of Anopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial., Methods: Anopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3-5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species, kdr and Ace-1 mutations., Results: Anopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereas Anopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested were Anopheles coluzzii . The frequencies of Ace-1R mutation in all Anopheles coluzzii populations tested were low (3-27%)., Conclusions: Organophosphates (malathion and pirimiphos-methyl) have maintained their efficacy against Anopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates against Anopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Dossou C et al.)

The use of black soldier fly (BSF) larvae and frass in agriculture can make an important contribution to food and nutrition security. However, it is important to understand whether consumers are willing to consume food products resulting from the use of BSF larvae as animal feed or BSF frass as fertilizer. This study employed the stated preference approach as food products produced using BSF larvae and frass are not currently available on the market. Questionnaires were administered to a total of 4412 consumers in Ghana (1360), Mali (1603), and Niger (1449). The results show that the vast majority of respondents are willing to consume vegetables (88%) produced using BSF frass and meat (87%) produced using animal feed made of BSF larvae. A smaller percentage of respondents are even willing to pay USD 1.32 and USD 1.7 more if the base price of BSF-based products were USD 5 per kg. Age, gender, education, and country positively influenced the respondents' willingness to consume food produced using BSF products. In contrast, neighborhood status, income, and household size are inversely related to the respondents' willingness to pay for and consume these products. Our findings are, therefore, important to scaling up BSF technologies in the region.

Background: This study aimed to investigate the relationship between the physicochemical characteristics of An. gambiae s.s. and An. coluzzii breeding sites, the susceptibility profiles to commonly used insecticides in public health, and the underlying insecticide resistance mechanisms., Methods: Anopheles breeding sites surveys were conducted in Cotonou and Natitingou in September 2020, January and August 2021. Physicochemical properties and bacterial loads were determined in individual breeding sites. The WHO susceptibility assays were carried out using the female of the emerging adult mosquitoes. Anopheles species were identified through PCR techniques. Kdr L1014F/S, N1575Y and G119S mutations were investigated using TaqMan genotyping assays., Results: Molecular analysis showed that all mosquitoes analyzed in Cotonou were Anopheles coluzzii, while those of Natitingou were Anopheles gambiae s.s. Fecal coliforms were identified as playing a role in this distribution through their significant influence on the presence of An. coluzzii larvae. WHO susceptibility assay indicated a high level of resistance to deltamethrin in the two cities. The resistance levels to deltamethrin were higher in Cotonou (X 2  = 31.689; DF = 1; P < 0.0001). There was a suspected resistance to bendiocarb in Cotonou, whereas the mosquito population in Natitingou was resistant. The kdr L1014F mutation was highly observed in both mosquito populations (frequence: 86-91%), while the Ace-1 mutation was found in a small proportion of mosquitoes. In Cotonou, salinity was the only recorded physicochemical parameter that significantly correlated with the resistance of Anopheles mosquitoes to deltamethrin (P < 0.05). In Natitingou, significant correlations were observed between the allelic frequencies of the kdr L1014F mutation and pH, conductivity, and TDS., Conclusion: These results indicate a high level of pyrethroid resistance in the anopheles populations of both Cotonou and Natitingou. Moreover, this study report the involvement of abiotic factors influencing Anopheles susceptibility profile., (© 2024. The Author(s).)

Malaria vector surveillance tools often incorporate features of hosts that are attractive to blood-seeking females. The recently developed host decoy trap (HDT) combines visual, thermal, and olfactory stimuli associated with human hosts and has shown great efficacy in terms of collecting malaria vectors. Synthetic odors and yeast-produced carbon dioxide (CO2) could prove useful by mimicking the human odors currently used in HDTs and provide standardized and easy-to-use olfactory attractants. The objective of this study was to test the attractiveness of various olfactory attractant cues in HDTs to capture malaria vectors. We compared 4 different odor treatments in outdoor field settings in southern Benin and western Burkina Faso: the standard HDT using a human, HDT with yeast-produced CO2, HDT with an artificial odor blend, and HDT with yeast-produced CO2 plus artificial odor blend. In both experimental sites, the standard HDT that incorporated a real human produced the greatest catch of Anopheles gambiae s.l (Diptera: Culicidae). The alternatives tested were still effective at collecting target vector species, although the most effective included CO2, either alone (Benin) or in combination with synthetic odor (Burkina Faso). The trap using synthetic human odor alone caught the fewest An. gambiae s.l. compared to the other baited traps. Both Anopheles coluzzii and Anopheles gambiae were caught by each trap, with a predominance of An. coluzzii. Synthetic baits could, therefore, represent a more standardized and easier-to-deploy approach than using real human odor baits for a robust vector monitoring strategy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The primary reason for the failure of malaria vector control across endemic regions is the widespread insecticide resistance observed in Anopheles vectors. The most dominant African vectors of malaria parasites are Anopheles gambiae and Anopheles funestus mosquitoes. These species often exhibit divergent behaviours and adaptive changes underscoring the importance of deploying active and effective measures in their control. Unlike An. gambiae, An. funestus mosquitoes are poorly studied in Benin Republic. However, recent reports indicated that An. funestus can adapt and colonize various ecological niches owing to its resistance against insecticides and adaptation to changing breeding habitats. Unfortunately, scientific investigations on the contribution of An. funestus to malaria transmission, their susceptibility to insecticide and resistance mechanism developed are currently insufficient for the design of better control strategies. In an attempt to gather valuable information on An. funestus, the present review examines the progress made on this malaria vector species in Benin Republic and highlights future research perspectives on insecticide resistance profiles and related mechanisms, as well as new potential control strategies against An. funestus. Literature analysis revealed that An. funestus is distributed all over the country, although present in low density compared to other dominant malaria vectors. Interestingly, An. funestus is being found in abundance during the dry seasons, suggesting an adaptation to desiccation. Among the An. funestus group, only An. funestus sensu stricto (s.s.) and Anopheles leesoni were found in the country with An. funestus s.s. being the most abundant species. Furthermore, An. funestus s.s. is the only one species in the group contributing to malaria transmission and have adapted biting times that allow them to bite at dawn. In addition, across the country, An. funestus were found resistant to pyrethroid insecticides used for bed nets impregnation and also resistant to bendiocarb which is currently being introduced in indoor residual spraying formulation in malaria endemic regions. All these findings highlight the challenges faced in controlling this malaria vector. Therefore, advancing the knowledge of vectorial competence of An. funestus, understanding the dynamics of insecticide resistance in this malaria vector, and exploring alternative vector control measures, are critical for sustainable malaria control efforts in Benin Republic., (© 2023. The Author(s).)

Background & Rationale: Malaria is a major health problem in Benin where it is the main cause of morbidity and mortality, particularly among children under 5 and pregnant women. Although the vast majority of malaria cases occurs in rural and agricultural areas and are often associated with development projects, very few interventions target the agro-ecosystem. In Benin, irrigated rice growing is expanding to meet the increasing demand of the population. However, continuous flooding and tillage systems induce the development and proliferation of malaria and other diseases vectors. Intermittent flooding of rice plots and minimal tillage can reduce significantly the proliferation of mosquitoes including Anopheles in rice fields. However, the dissemination and implementation of these agricultural practices require community-wide action for greater effectiveness. As part of strengthening the capacity of farming communities in the fight against malaria vectors, the "Farmer Field School" appears to be an innovative approach. This learning by doing method promotes interactions between groups of producers to disseminate proven technologies. This study aims to disseminate among rice producers the agricultural practices of intermittent flooding and minimal tillage, likely to reduce the proliferation of malaria vectors in the rice fields., Materials & Methods: This study was carried out in the rice-growing perimeter of Malanville, Benin (11° 52' 5" North, 3° 22' 59" East) which covers an area of 516 hectares. Farmer Field Schools were set up after a basic survey at producer level. This survey was carried out through in-depth interview, focus group discussions and direct field observation with producers. Focus groups discussions and interviews made it possible to understand the perception of rice farmers on the link between rice production and the transmission of malaria. In order to disseminate new agricultural practices such as intermittent flooding and minimal tillage among producers, twelve plots have been set up. Farmer Field Schools were monitored weekly with rice producers accompanied by a facilitator and a medical entomologist (learning facilitator or moderator) helping the farmers with the collection and identification of mosquito larvae. According to the different stages of rice development (transplanting, tillering, maturation), the mosquito larvae were collected in the test and control plots from 10 a.m. to 2 p.m. by the dipping method. Then the water in the test compartments (intermittent flooding) was emptied. A cycle of 7 days of flooding and 2 days of drying was carried out for intermittent flooding. Mosquito larvae were identified morphologically using the identification key and Anopheles genus larvae were isolated in plastic cups. The impacts of intermittent flooding and minimum tillage in reducing breeding sites and larval densities were established by determining and comparing the larval densities of mosquitoes and of Anopheles between the test and control plots., Results: Direct observations in the field allowed to identify three tillage systems, which include the use of tiller (28%), plow (66%) and hoe (6%) on the rice field. Continuous flooding was the only irrigation system used by farmers. The water used for irrigation comes either from boreholes installed individually or from the Niger River. The volume of water used varies with the seasons, the size of the farms and the variety of rice grown. Farmers observe that the nuisance of mosquitoes increases during the rice production period with an outbreak of malaria cases, especially among children, which leads to crowds in health centers. The preventive measures against malaria among farmers are the use of impregnated mosquito nets distributed free of charge by the national program against malaria, and of insecticide bombs or spirals. Considering the development stages of the rice, the larval densities varied according to the treatments. Overall, minimal tillage applied to intermittent flooding significantly reduced the density of mosquito larvae of all species. The reduction rates were 10.5, 5.4 and 2.5 during transplanting, tillering and maturation, respectively. Considering only the Anopheles larvae, minimal tillage applied to intermittent flooding reduced their density by 16, 5.5 and 4 respectively during transplanting, tillering and maturation., Discussion/conclusion: The rice-growing area of Malanville has many favorable conditions for rice production, including the presence of water supply sources such as the Niger River located near the rice field and numerous boreholes. The availability of water pockets for mosquito breeding during irrigation appeared to contribute to the extension of malaria transmission. The present study showed that the intermittent flooding coupled with minimal tillage could reduce the proliferation of malaria vectors. The results suggested that with technical support to farmers through the "Farmer Field School", the malaria incidence could be reduced in the farming community., (Copyright © 2023 SFMTSI.)

Background: The COVID-19 pandemic and its damages have severely impacted the global healthcare system even in countries with the best systems. In sub-Saharan Africa (SSA), it could worsen the malaria situation in endemic countries such as Benin. This study was conducted to describe the potential effects of the pandemic on urban dwellers attitudes, prevention and treatment against malaria in four major cities of Benin., Methods: A cross-sectional questionnaire survey was conducted in Cotonou, Bohicon, Parakou and Natitingou, four urban cities of Benin. A total of 800 randomly selected households were interviewed. The questionnaire consisted of four parts: sociodemographic characteristics, knowledge, attitude, and practice. Descriptive statistics and binomial logistic regression analysis were used in the statistical analysis., Results: More than 90% of the participants interviewed had a good level of knowledge about the transmission and prevention of malaria in the cities surveyed. In contrast, low proportions of participants reported visiting health-care facilities when they suspected malaria. Compared to the proportions observed at Parakou and Natitingou, the low proportion of participants was statistically significant at Cotonou (Parakou: X 2  = 31.345, df = 1, P < 0.0001; Natitingou: X 2  = 17.471, df = 1, P < 0.0001). Among the reasons for not seeking care, these related to COVID-19 were the most mentioned. Moreover, the good education level of the participants was one of the factors associated with the non-use of healthcare facilities due to over-knowledgeable about Covid-19, which might have increased the fear to go to the health facilities. Finally, high proportions of self-medication practice were mentioned with high use of malaria drugs to treat both malaria and to protect against COVID-19., Conclusions: The data show a negative impact of COVID-19 on visits to healthcare facilities for malarial treatment and malaria drugs usage by the population. It is, therefore, necessary to rebuild malaria programmes by integrating measures adapted to health crises such as the COVID-19 pandemic., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Arthropod-borne viruses (Arboviruses) replicate in vertebrates and invertebrates and are mainly transmitted by mosquitoes. Between 2000 and 2021, several arbovirus outbreaks were recorded in African countries, including dengue, yellow fever, Chikungunya, Zika, and O'nyong nyong. Most often, the causes and factors involved in these outbreaks are unknown. We aimed to understand current knowledge regarding factors responsible for the persistent transmission and emergence of mosquito-borne arboviruses in Africa and to identify critical research gaps important for preventing future outbreaks. We used a systematic literature review between 2020 and 2021, to show that the main identified factors favoring the arbovirus outbreak in Africa are low vaccination coverage, high density and diversity of competent mosquitoes, insecticide resistance of mosquito vectors, and a scarcity of data on arboviruses. Further studies on arboviruses may include studies of competence to viral strains and the susceptibility of mosquito vectors to insecticides. Because of the detrimental effects of insecticides on human health and the environment, viral paratransgenesis and other biological control methods should be explored as alternatives or as supplements to insecticides., Graphical Abstract: Illustration of factors identified for promoting the transmission of arbovirus in Africa. The main factors are the lack of drugs and vaccines, low coverage of vaccination when a vaccine exists, competence of mosquitoes to viruses, diversity and high density of vectors. Climate change, urbanization, deforestation and agricultural practices, lead to a richness and high density of vectors., Competing Interests: Conflict of interestThe 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., (© The Author(s), under exclusive licence to Indian Virological Society 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F0) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.

Background: The implementation of anti-larval strategies in the fight against malaria vectors requires fundamental knowledge of their oviposition sites. The aim of this study was to assess the spatial and temporal distribution of Anopheles breeding sites as well as the influence of abiotic and biotic factors on the proliferation of larvae in urban and non-urban areas of Benin., Methods: Sampling of Anopheles larvae was carried out during the rainy and dry seasons in urbanized and non-urbanized areas of the cities of Cotonou, Bohicon, Parakou, and Natitingou in Benin. The Anopheles larval breeding sites were georeferenced and characterized by their nature, type, physicochemical (pH, temperature, dissolved oxygen, conductivity, turbidity, salinity) and biological attributes (larval density and coliform density)., Results: A total of 198 positive breeding sites for Anopheles larvae were identified, comprising 163 (82.3%) in the rainy season and 35 (17.7%) in the dry season. Out of these larval habitats, 61.9% were located in urbanized areas, and were predominantly puddles. Principal component analysis revealed a high positive correlation of larval density with temperature and dissolved oxygen, and with salinity in the coastal zone. In addition, cross-sectional analysis of the microbiological results with larval density showed a significant negative correlation between larval productivity and faecal coliform load., Conclusions: This study indicated the presence of multiple larval habitats of Anopheles in the urban areas which were created through human activities, and associations between larval density and intrinsic factors of the habitats such as temperature, dissolved oxygen and faecal coliform load. This type of information may be useful for the implementation of appropriate control strategies in urban areas, including regulation of the human activities that lead to the creation of breeding sites, proper environmental management and targeted larvicidal use., (© 2022. The Author(s).)

Nickel(II) tetrasulfonated phthalocyanine (p-NiTSPc)-modified carbon fiber microelectrode (CFME) was used for the first time to investigate the electrochemical quantification of diuron in an agrochemical formulation. The surface morphology and elementary analysis of unmodified carbon fiber microelectrode (CFME) and p-NiTSPc-CFME were performed using atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX), respectively. Cyclic voltammetry (CV) was used to investigate the electrochemical behaviour of diuron, while square wave voltammetry (SWV) was used for quantitative analysis of diuron. Upon variation of some key analytical parameters, a calibration curve was plotted in the concentration range from 21.450 to 150.150  μ M, leading to a detection limit (DL) of 8.030  μ M mg/L (3.3 σ /m) and a limit of quantification (LQ) of 20.647  μ M mg/L. The fabricated p-NiTSPc-CFME was successfully applied for quality control in a commercialized formulation of diuron. The standard additional method was used, and the recovery rate of diuron was found to be 98.4%., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2022 Serge Mbokou Foukmeniok et al.)

Background: Rice fields in Africa are major breeding sites for malaria vectors. However, when reviewed in the 1990s, in settings where transmission was relatively intense, there was no tendency for malaria indices to be higher in villages with irrigated rice fields than in those without. Subsequently, intervention coverage in sub-Saharan Africa has been massively scaled up and malaria infection prevalence has halved. We re-examined this rice-malaria relationship to assess whether, with lower malaria transmission, malaria risk is greater in rice-growing than in non-rice-growing areas., Methods: For this systematic review and meta-analysis, we searched EMBASE, Global Health, PubMed, Scopus, and Web of Science to identify observational studies published between Jan 1, 1900, and Sept 18, 2020. Studies were considered eligible if they were observational studies (cross-sectional, case-control, or cohort) comparing epidemiological or entomological outcomes of interest between people living in rice-growing and non-rice-growing rural communities in sub-Saharan Africa. Studies with pregnant women, displaced people, and military personnel as participants were excluded because they were considered not representative of a typical community. Data were extracted with use of a standardised data extraction form. The primary outcomes were parasite prevalence (P falciparum parasite rate age-standardised to 2-10-year-olds, calculated from total numbers of participants and number of infections [confirmed by microscopy or rapid diagnostic test] in each group) and clinical malaria incidence (number of diagnoses [fever with Plasmodium parasitaemia confirmed by microscopy or rapid diagnostic test] per 1000 person-days in each group). We did random-effects meta-analyses to estimate the pooled risk ratio (RR) for malaria parasite prevalence and incidence rate ratio (IRR) for clinical malaria in rice-growing versus non-rice-growing villages. RRs were compared in studies conducted before and after 2003 (chosen to mark the start of the mass scale-up of antimalaria interventions). This study is registered with PROSPERO (CRD42020204936)., Findings: Of the 2913 unique studies identified and screened, 53 studies (including 113 160 participants across 14 African countries) were eligible for inclusion. In studies done before 2003, malaria parasite prevalence was not significantly different in rice-growing versus non-rice-growing villages (pooled RR 0·82 [95% CI 0·63-1·06]; 16 studies, 99 574 participants); however, in post-2003 studies, prevalence was significantly higher in rice-growing versus non-rice growing villages (1·73 [1·01-2·96]; seven studies, 14 002 participants). Clinical malaria incidence was not associated with residence in rice-growing versus non-rice-growing areas (IRR 0·75 [95% CI 0·47-1·18], four studies, 77 890). Potential limitations of this study include its basis on observational studies (with evidence quality rated as very low according to the GRADE approach), as well as its omission for the effects of seasonality and type of rice being cultivated. Risk of bias and inconsistencies was relatively serious, with I 2 greater than 90% indicating considerable heterogeneity., Interpretation: Irrigated rice-growing communities in sub-Saharan Africa are exposed to greater malaria risk, as well as more mosquitoes. As increasing rice production and eliminating malaria are two major development goals in Africa, there is an urgent need to improve methods for growing rice without producing mosquitoes., Funding: Wellcome Trust Our Planet Our Health programme, CGIAR Agriculture for Nutrition and Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Onchocerciasis elimination is within reach in many countries but requires enhanced surveillance of the Simulium vectors of Onchocerca volvulus. Collection of sufficient numbers of adult Simulium to detect infective O. volvulus larvae is hindered by limited sampling tools for these flies. Here, we tested for the first time the Host Decoy Trap (HDT), an exposure free method previously developed for Anopheles vectors of malaria parasites, as a potential sampling tool for adult Simulium. In three replicates of a randomized Latin square experimental design, the HDT was compared to Human Landing Catches (HLC) and the Esperanza Window Trap (EWT). A total of 8,531 adult S. damnosum sensu lato blackflies (S. squamosum group) were found in catches from the three different trapping methods. The HDT (mean catch 533 ± 111) caught significantly more S. squamosum than the EWT (mean catch 9.1 ± 2.2), a nearly 60-fold difference. There was no significant difference between the HLC (mean catch 385.6 ± 80.9) and the HDT. Larvae indistinguishable from those of O. volvulus were dissected from 2.86% of HDT samples (n = 70) and 0.35% of HLC samples (n = 285); a single infective third-stage larvae (L3) was  found during dissection of a sample from the HDT. Owing to its very high capture rate, which was comparable to the HLC and significantly greater than EWT, alongside the presence of infected flies in its catch, the HDT represents a potentially valuable new tool for blackfly collection in elimination settings, where thousands of flies are needed for parasite screening., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments., Methods: Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS2404, IS2606, KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19)., Results: MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities., Conclusions: VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.

Background: Insecticide-based vector control is responsible for reducing malaria mortality and morbidity. Its success depends on a better knowledge of the vector, its distribution, and resistance status to the insecticides used. In this paper, we assessed Anopheles gambiae sensu lato (A gambiae s.l.) population resistance to pyrethroids in different ecological settings., Methods: The World Health Organization standard bioassay test was used to assess F0A gambiae s.l. susceptibility to pyrethroids. Biochemical Synergist assays were conducted with piperonyl butoxide (PBO), S,S,S-tributyl phosphotritioate, and diethyl maleate. L1014F, L1014S, and N1575Y knockdown resistance (kdr) mutations were investigated using TaqMan genotyping., Results: Anopheles gambiae sensu lato was composed of Anopheles arabienisis, Anopheles coluzzii, and A gambiae in all study sites. Anopheles gambiae sensu lato showed a strong phenotypic resistance to deltamethrin and permethrin in all sites (13% to 41% mortality). In many sites, pre-exposure to synergists partially improved the mortality rate suggesting the presence of detoxifying enzymes. The 3 kdr (L1014F, L1014S, and N1575Y) mutations were found, with a predominance of L1014F, in all species., Conclusions: Multiple resistance mechanisms to pyrethroids were observed in A gambiae s.l. in Mali. The PBO provided a better partial restoration of susceptibility to pyrethroids, suggesting that the efficacy of long-lasting insecticidal nets may be improved with PBO., (© World Health Organization, 2021. All rights reserved. The World Health Organization has granted the Publisher permission for the reproduction of this article.)

Background: Understanding the molecular basis of insecticide resistance in mosquito, such as Anopheles funestus, is an important step in developing strategies to mitigate the resistance problem. This study aims to assess the role of the GSTe2 gene in DDT resistance and determine the genetic diversity of this gene in An. funestus., Methods: Gene expression analysis was performed using microarrays and PCR while the potential mutation associated with resistance was determined using sequencing., Results: Low expression level of GSTe2 gene was recorded in Burkina-Faso samples with a fold change of 3.3 while high expression (FC 35.6) was recorded in southern Benin in Pahou (FC 35.6) and Kpome (FC 13.3). The sequencing of GSTe2 gene in six localities showed that L119F-GSTe2 mutation is almost getting fixed in highly DDT-resistant Benin (Pahou, Kpome, Doukonta) and Nigeria (Akaka Remo) mosquitoes with a low mutation rate observed in Tanongou (Benin) and Burkina-Faso mosquitoes., Conclusion: This study shows the key role of the GSTe2 gene in DDT resistant An. funestus in Benin. Polymorphism analysis of this gene across Benin revealed possible barriers to gene flow, which could impact the design and implementation of resistance management strategies in the country.

Background: Irrigation systems have been identified as one of the factors promoting malaria disease around agricultural farms in sub-Saharan Africa. However, if improved water management strategy is adopted during rice cultivation, it may help to reduce malaria cases among human population living around rice fields. This study aimed to assess the impact of the different irrigation practices on malaria transmission, as well as to evaluate the water management system that will best mitigate malaria transmission in Malanville, Benin., Methods: Knowledge, Attitude and Practice (KAP) study was conducted on 104 households staying on and around the rice fields in Malanville. The study focused on the frequency of mosquito bites and preventive measures against malaria as well as soil preparation and rice planting methods. Mosquito larvae density was assessed in different water management system: continuous flooding (CF) or intermittent flooding (IF), deep tillage (DT) or minimal tillage (MT) and normal levelling (NL) or abnormal levelling (AL) in an experimental hut set-up. Larvae were collected using dipping methods and their density was determined., Results: Three tillage systems, which include the use of tiller, plow and hoe, were identified on the rice field. Continuous flooding was the only irrigation system used by farmers. Retrospective data from Malanville Health Centre revealed higher malaria cases during rice production season, which was also confirmed by field participants. The density of Anopheles larvae was reduced by 80.8%, 30.8% and 40.7% (P = 0.000) during transplanting, tillering and maturation periods, respectively with intermittent flooding compared to continuous flooding. In addition, a clear reduction of larva density was observed with both intermittent flooding systems applied to minimal tillage (MT + IF + NL) and intermittent flooding applied to deep tillage (DT + IF + AL), showing that intermittent flooding could reduce the abundance of malaria vector in rice fields., Conclusion: Recommending intermittent flooding technology for rice cultivation may not only be useful for water management but could also be an intentional strategy to control mosquitoes vector-borne diseases around rice farms.

Background: Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated., Methods: Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method., Results: Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance., Conclusions: The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

Vector control strategies recommended by the World Health Organization are threatened by resistance of Anopheles mosquitoes to insecticides. Information on the distribution of resistant genotypes of malaria vectors is increasingly needed to address the problem. Ten years of published and unpublished data on malaria vector susceptibility/resistance and resistance genes have been collected across Togo. Relationships between the spatial distribution of resistance status and environmental, socio-economic, and landscape features were tested using randomization tests, and calculating Spearman rank and Pearson correlation coefficients between mosquito mortality and different gridded values. Anopheles gambiae sensu lato was resistant to DDT, pyrethroids, and the majority of carbamates and organophosphates. Three sibling species were found (i.e., An. gambiae, Anopheles coluzzii, and Anopheles arabiensis) with four resistance genes, including kdr (L1014F, L1014S, and N1575Y) and ace1 (G119S). The most frequent resistance gene was L1014F. Overall, no association was found between the susceptibility/resistance status and environmental features, suggesting that evolution of resistance may be most closely related to extreme selection from local insecticide use. Nevertheless, further research is necessary for firm conclusions about this lack of association, and the potential role of landscape characteristics such as presence of crops and percentage of tree cover., (© The Author(s) 2020. 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 long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are major malaria vector control strategies in Mali. The success of control strategies depends on a better understanding of the status of malaria vectors with respect to the insecticides used. In this study we evaluate the level of resistance of Anopheles gambiae (sensu lato) to bendiocarb and the molecular mechanism that underlies it., Methods: Larvae of An. gambiae (s.l.) were collected from breeding habitats encountered in the three study sites and bioassayed with bendiocarb. The ace-1 target site substitution G119S was genotyped using a TaqMan assay., Results: The three species of the An. gambiae complex in Mali, i.e. An. arabiensis, An. coluzzii and An. gambiae (s.s.) were found in sympatry in the three surveyed localities with different frequencies. We observed a resistance and suspicious resistance of the three species to bendiocarb with a mortality rate ranging from 37% to 86%. The allelic frequency of the G119S mutation was higher in An. gambiae (s.s.) compared to the other two species; 42.86%, 25.61% and 16.67% respectively in Dangassa, Koula, and Karadié. The allelic frequency of G119S in An. coluzzii ranged from 4.5% to 8.33% and from 1.43% to 21.15% for An. arabiensis. After exposure to bendiocarb, the G119S mutation was found only in survivors. The survival of Anopheles gambiae (s.l) populations from the three surveyed localities was associated with the presence of the mutation., Conclusions: The study highlights the implication of G119S mutation in bendiocarb resistance in An. gambiae (s.s.), An. arabiensis and An. coluzzii populations from the three surveyed localities.

The use of agrochemicals in vegetable production could influence the selection for insecticide resistance in malaria vectors. Unfortunately, there is a dearth of information on the potential contribution of agrochemicals to insecticide resistance in Anopheles mosquitoes breeding on vegetable farms in southern Benin. A Knowledge, Attitudes and Practices study was conducted with 75 vegetable farmers from Houeyiho and Seme to determine the main agrochemicals used in vegetable production, and the concentration and frequency of application, among other details. Mosquitoes and breeding water were sampled from the farms for analysis. Bioassays were conducted on mosquitoes, while breeding water was screened for heavy metal and pesticide residue contamination. Lambda-cyhalothrin was the main insecticide (97.5%) used by farmers, and Anopheles coluzzii was the main mosquito identified. This mosquito species was resistant (30-63% mortality rate) to λ-cyhalothrin. It was also observed that 16.7% of the examined breeding sites were contaminated with λ-cyhalothrin residues. Furthermore, copper contamination detected in mosquito breeding sites showed a positive correlation (r = 0.81; P = 0.0017) with mosquito resistance to λ-cyhalothrin. The presence of copper in λ-cyhalothrin-free breeding sites, where mosquitoes have developed resistance to λ-cyhalothrin, suggests the involvement of copper in the insecticide resistance of malaria vectors; this, however, needs further investigation.

Termites are widely used as a food resource, particularly in Africa and Asia. Markets for insects as food are also expanding worldwide. To inform the development of insect-based foods, we analysed selected minerals (Fe-Mn-Zn-Cu-Mg) in wild-harvested and commercially available termites. Mineral values were compared to selected commercially available insects. Alate termites, of the genera Macrotermes and Odontotermes, showed remarkably high manganese (Mn) content (292-515 mg/100 gdw), roughly 50-100 times the concentrations detected in other insects. Other mineral elements occur at moderate concentrations in all insects examined. On further examination, the Mn is located primarily in the abdomens of the Macrotermes subhyalinus; with scanning electron microscopy revealing small spherical structures highly enriched for Mn. We identify the fungus comb, of Macrotermes subhyanus, as a potential biological source of the high Mn concentrations. Consuming even small quantities of termite alates could exceed current upper recommended intakes for Mn in both adults and children. Given the widespread use of termites as food, a better understanding the sources, distribution and bio-availability of these high Mn concentrations in termite alates is needed.

Helicoverpa armigera is an indigenous species in Africa and has been reported in the destruction of several crops in Benin. Management of H. armigera pest is mainly focused on the use of synthetic pyrethroids, which may contribute to resistance selection. This study aimed to screen the susceptibility pattern of field populations of H. armigera to deltamethrin in Benin. Relevant information on the type of pesticides used by farmers were gathered through surveys. Collected samples of Helicoverpa (F 0 ) were reared to F 1 . F 0 were subjected to morphological speciation followed by a confirmation using restriction fragment length polymorphism coupled with a polymerase chain reaction (RFLP-PCR). F 1 (larvae) were used for insecticide susceptibility with deltamethrin alone and in the presence of the P450 inhibitor Piperonyl Butoxide (PBO). Deltamethrin and lambda-cyhalothrin were the most used pyrethroids in tomato and cotton farms respectively. All field-sampled Helicoverpa were found to be H. armigera . Susceptibility assays of H. armigera to deltamethrin revealed a high resistance pattern in cowpea (resistance factor (RF) = 2340), cotton (RF varying from 12 to 516) and tomato (RF=85) farms which is a concern for the control of this major polyphagous agricultural pest. There was a significant increase of mortality when deltamethrin insecticide was combined with piperonyl butoxide (PBO), suggesting the possible involvement of detoxification enzymes such as oxidase. This study highlights the presence of P450 induced metabolic resistance in H. armigera populations from diverse cropping systems in Benin. The recorded high levels of deltamethrin resistance in H. armigera is a concern for the control of this major agricultural pest in Benin as the country is currently embarking into economical expansion of cotton, vegetables and grain-legumes cropping systems., Competing Interests: The authors declare no conflict of interest.

Metabolic resistance to insecticides such as pyrethroids in mosquito vectors threatens control of malaria in Africa. Unless it is managed, recent gains in reducing malaria transmission could be lost. To improve monitoring and assess the impact of insecticide resistance on malaria control interventions, we elucidated the molecular basis of pyrethroid resistance in the major African malaria vector, Anopheles funestus We showed that a single cytochrome P450 allele ( CYP6P9a &#95;R) in A. funestus reduced the efficacy of insecticide-treated bednets for preventing transmission of malaria in southern Africa. Expression of key insecticide resistance genes was detected in populations of this mosquito vector throughout Africa but varied according to the region. Signatures of selection and adaptive evolutionary traits including structural polymorphisms and cis-regulatory transcription factor binding sites were detected with evidence of selection due to the scale-up of insecticide-treated bednet use. A cis-regulatory polymorphism driving the overexpression of the major resistance gene CYP6P9a allowed us to design a DNA-based assay for cytochrome P450-mediated resistance to pyrethroid insecticides. Using this assay, we tracked the spread of pyrethroid resistance and found that it was almost fixed in mosquitoes from southern Africa but was absent from mosquitoes collected elsewhere in Africa. Furthermore, a field study in experimental huts in Cameroon demonstrated that mosquitoes carrying the resistance CYP6P9a&#95;R allele survived and succeeded in blood feeding more often than did mosquitoes that lacked this allele. Our findings highlight the need to introduce a new generation of insecticide-treated bednets for malaria control that do not rely on pyrethroid insecticides., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. In order to implement suitable insecticide resistance management strategies, it is necessary to understand the underlying mechanisms involved. To achieve this, the molecular basis of permethrin and DDT resistance in the principal malaria vector, Anopheles funestus from inland Benin (Kpome), was investigated., Results: Here, using a microarray-based genome-wide transcription and qRT-PCR analysis, we showed that metabolic resistance mechanisms through over-expression of cytochrome P450 and glutathione S-transferase genes (GSTs) are a major contributor to DDT and permethrin resistance in Anopheles funestus from Kpome. The GSTe2 gene was the most upregulated detoxification gene in both DDT- [fold-change (FC: 16.0)] and permethrin-resistant (FC: 18.1) mosquitoes suggesting that upregulation of this gene could contribute to DDT resistance and cross-resistance to permethrin. CYP6P9a and CYP6P9b genes that have been previously associated with pyrethroid resistance were also significantly overexpressed with FC 5.4 and 4.8, respectively, in a permethrin resistant population. Noticeably, the GSTs, GSTd1-5 and GSTd3, were more upregulated in DDT-resistant than in permethrin-resistant Anopheles funestus suggesting these genes are more implicated in DDT resistance. The absence of the L1014F or L1014S kdr mutations in the voltage-gated sodium channel gene coupled with the lack of directional selection at the gene further supported that knockdown resistance plays little role in this resistance., Conclusions: The major role played by metabolic resistance to pyrethroids in this An. funestus population in Benin suggests that using novel control tools combining the P450 synergist piperonyl butoxide (PBO), such as PBO-based bednets, could help manage the growing pyrethroid resistance in this malaria vector in Benin.

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

Background: The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions., Methodology/principal Findings: We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions., Conclusion/significance: This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway., Competing Interests: The authors have declared that no competing interests exist.

Background: Insecticides resistance in Anopheles mosquitoes limits Long-Lasting Insecticidal Nets (LLIN) used for malaria control in Africa, especially Benin. This study aimed to evaluate the bio-efficacy of current LLINs in an area where An. funestus s.l. and An. gambiae have developed multi-resistance to insecticides, and to assess in experimental huts the performance of a mixed combination of pyrethroids and piperonyl butoxide (PBO) treated nets on these resistant mosquitoes. Methods: The study was conducted at Kpomè, Southern Benin. The bio-efficacy of LLINs against An. funestus and An. gambiae was assessed using the World Health Organization (WHO) cone and tunnel tests. A released/recapture experiment following WHO procedures was conducted to compare the efficacy of conventional LLINs treated with pyrethroids only and LLINs with combinations of pyrethroids and PBO. Prior to huts trials, we confirmed the level of insecticide and PBO residues in tested nets using high performance liquid chromatography (HPLC). Results: Conventional LLINs (Type 2 and Type 4) have the lowest effect against local multi-resistant An. funestus s.s. and An. coluzzii populations from Kpomè. Conversely, when LLINs containing mixtures of pyrethroids and PBO (Type 1 and Type 3) were introduced in trial huts, we recorded a greater effect against the two mosquito populations (P < 0.0001). Tunnel test with An. funestus s.s. revealed mortalities of over 80% with this new generation of LLINs (Type 1 and Type 3),while conventional LLINs produced 65.53 ± 8.33% mortalities for Type 2 and 71.25 ±7.92% mortalities for Type 4. Similarly, mortalities ranging from 77 to 87% were recorded with the local populations of An. coluzzii . Conclusion: This study suggests the reduced efficacy of conventional LLINs (Pyrethroids alone) currently distributed in Benin communities where Anopheles populations have developed multi-insecticide resistance. The new generation nets (pyrethroids+PBO) proved to be more effective on multi-resistant populations of mosquitoes., Competing Interests: No competing interests were disclosed.

The management of the fall armyworm Spodoptera frugiperda in maize field necessitates the use a big quantities of insecticides and sometimes the use of multiple types and formulations of chemicals. The use of insecticides in crops is associated with environmental risks and health hazards to both producers and consumers. This study was designed to evaluate the residue of 11 insecticides that were used to control high population of the fall armyworm in maize field in Mokwa, Nigeria. Maize and soil samples were collected from an experimental field to investigate the residue level using high performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) analysis techniques. Results revealed the presence of five insecticide compounds (Cypermethrin, Deltamethrin, Lambda-Cyhalothrin, Permethrin, and Chorpyrifos) in soil samples with possible adverse effects on soil born organisms and other non-targeted species. In contrast, no residue was found in maize stems and seeds. From these results, we conclude that the treated maize remains safe for consumption and the producers may not get any serious risk of contamination from the chemical control of the fall armyworm., Competing Interests: The authors declare no conflict of interest.

Background: To optimize the success of insecticide-based malaria control intervention, knowledge of the distribution of Anopheles gambiae species and insecticide resistance mechanisms is necessary. This paper reported an updated data on pyrethroids/DDT resistance in the An. gambiae s.l population from Togo.  Methods: From December 2013 to April 2015, females of indoor-resting An. gambiae s.l were captured in three locations belonging to three different ecological zones. Resistance to DDT, permethrin and deltamethrin was screened in F1 progeny of collected mosquitoes using WHO susceptibility tests. The identification of species of An. gambiae complex and the detection of kdr and ace.1 R allele were carried out using DNA-based molecular techniques. Results: An. gambiae from Kovié and Nangbéto were highly resistant to DDT and permethrin with mortalities rate ranging from 0.83% to 1.58% for DDT and zero to 8.54% for permethrin. Mosquitoes collected in Nangbéto displayed 81.53% mortality with deltamethrin. An. coluzzii and An. gambiae s.s were found in sympatry in Nangbéto and Mango . The allelic frequency of L1014F was high, ranging from 66 to 100% in both An. coluzzii and An. gambiae s.s . For the first time we detected the L1014S allele in both An. coluzzii and An. gambiae s.s. from Togo at the frequency ranging from 5% to 13% in all the sites. The kdr N1575Y was present at various frequencies in both species ranging from 10% to 45%. Both An. gambiae s.s. and An. coluzzii shared the ace1 R mutation in all investigated sites with allelic frequency ranging from 4% to 16%. Conclusion: These results showed that multiple mutations are involved in insecticides resistance in An. gambiae populations from Togo including the kdr L1014F, L1014S, and N1575Y and ace.1 R G119S mutations., Competing Interests: No competing interests were disclosed.

The study of demographic processes involved in species diversification and evolution ultimately provides explanations for the complex distribution of biodiversity on earth, indicates regions important for the maintenance and generation of biodiversity, and identifies biological units important for conservation or medical consequence. African and forest biota have both received relatively little attention with regard to understanding their diversification, although one possible mechanism is that this has been driven by historical climate change. To investigate this, we implemented a standard population genetics approach along with Approximate Bayesian Computation, using sequence data from two exon-primed intron-crossing (EPIC) nuclear loci and mitochondrial cytochrome oxidase subunit I, to investigate the evolutionary history of five medically important and inherently forest dependent mosquito species of the genus Aedes . By testing different demographic hypotheses, we show that Aedes bromeliae and Aedes lilii fit the same model of lineage diversification, admixture, expansion, and recent population structure previously inferred for Aedes aegypti . In addition, analyses of population structure show that Aedes africanus has undergone lineage diversification and expansion while Aedes hansfordi has been impacted by population expansion within Uganda. This congruence in evolutionary history is likely to relate to historical climate-driven habitat change within Africa during the late Pleistocene and Holocene epoch. We find differences in the population structure of mosquitoes from Tanzania and Uganda compared to Benin and Uganda which could relate to differences in the historical connectivity of forests across the continent. Our findings emphasize the importance of recent climate change in the evolution of African forest biota.

Background:   The insecticide susceptibility status of Anopheles funestus, one of the main malaria vectors in the Afrotropical regions, remains under-studied due to the difficulty of working with this mosquito species. Collecting their larvae in natural breeding sites, rearing and maintaining them in normal laboratory conditions have been a difficult task. Forced-egg laying technique has been a very good tool to generate eggs from adult mosquitoes collected from the wild but rearing these eggs to obtain satisfying portion as adults has always been the problem. In this study, we optimized the development of mosquito species larvae under standard laboratory conditions for desired production of adult mosquitoes that can be useful for insecticide susceptibility tests. Methods:  A forced-egg laying technique was used to obtain eggs from gravid female Anopheles funestus collected from Kpome locality in Benin. Eggs were reared in three different water samples (water from the borehole,and two mineral water namely FIFA and Possotômè) and larvae were fed with TetraMin baby fish food. The physico-chemical parameters of the waters were investigated prior to use for egg incubation. Results: In contrast to mineral water that had no contamination, the borehole water source was contaminated with lead (2.5mg/L) and nitrate (118.8mg/L). Egg hatching rates ranged as 91.9 ± 4.4%, 89.1 ± 2.5% and 87.9 ± 2.6% in FIFA, Possotômè and borehole water respectively. High emergence of larvae to adult mosquitoes was recorded as in FIFA (74.3%) and Possotômè(79.5%) water. No adult mosquito was obtained from larvae reared in borehole water. Conclusions: This study gave insight on the water sources that could be good for rearing to mass produce An. funestus in the laboratory. More analysis with other local mineral water sources in our environments could be considered in the future, hopefully giving better outputs., Competing Interests: No competing interests were disclosed.

Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes ( CYP6P9a and CYP6P9b ) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies., (Copyright © 2017 Riveron et al.)

Background: Buruli ulcer (BU) continues to be a serious public health threat in wet tropical regions and the mode of transmission of its etiological agent, Mycobacterium ulcerans ( MU ), remains poorly understood. In this study, mosquito species collected in endemic villages in Benin were screened for the presence of MU . In addition, the ability of mosquitoes larvae to pick up MU from their environment and remain colonized through the larval developmental stages to the adult stage was investigated., Methods: 7,218 adults and larvae mosquitoes were sampled from endemic and nonendemic villages and screened for MU DNA targets ( IS2404 , IS2606 , and KR-B) using qPCR. Results. MU was not detected in any of the field collected samples. Additional studies of artificially infected larvae of Anopheles kisumu with MU strains revealed that mosquitoes larvae are able to ingest and host MU during L1, L2, L3, and L4 developmental stages. However, we noticed an absence of these bacteria at both pupae and adult stages, certainly revealing the low ability of infected or colonized mosquitoes to vertically transmit MU to their offspring., Conclusion: The overall findings highlight the low implication of mosquitoes as biological vectors in the transmission cycle of MU from the risk environments to humans.