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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.)

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.)

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).)

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.

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.

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.

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: 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.

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.

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.

Background: Knowledge on the spread and distribution of insecticide resistance in major malaria vectors such as Anopheles funestus is key to implement successful resistance management strategies across Africa. Here, by assessing the susceptibility status of an inland population of An. funestus Giles (Kpome) and investigating molecular basis of resistance, we show that multiple resistance and consistent plasmodium infection rate are present in Anopheles funestus populations from Kpome., Methods: The insecticide susceptibility level of collected Anopheles funestus was assessed. Synergist (PBO) was used to screen resistance mechanisms. The TaqMan technique was used for genotyping of insecticide resistant alleles and detecting plasmodium infection levels. The nested PCR was used to further assess the plasmodium infection rate., Results: The TaqMan analysis of plasmodial infections revealed an infection rate (18.2 %) of An. funestus in this locality. The WHO bioassays revealed a multiple phenotypic resistance profile for An. funestus in Kpome. This population is highly resistant to pyrethroids (permethrin and deltamethrin), organochlorines (DDT), and carbamates (bendiocarb). A reduced susceptibility was observed with dieldrin. Mortalities did not vary after pre-exposure to PBO for DDT indicating that cytochrome P450s play little role in DDT resistance in Kpome. In contrast, we noticed, a significant increase in mortalities when PBO was combined to permethrin suggesting the direct involvement of P450s in pyrethroid resistance. A high frequency of the L119F-GSTe2 DDT resistance marker was observed in the wild DDT resistant population (9 %RS and 91 %RR) whereas the A296S mutation was detected at a low frequency (1 %RS and 99 %SS)., Conclusion: The presence of multiple resistance in An. funestus populations in the inland locality of Kpome is established in this study as recently documented in the costal locality of Pahou. Data from both localities suggest that resistance could be widespread in Benin and this highlights the need for further studies to assess the geographical distribution of insecticide resistance across Benin and neighboring countries as well as a more comprehensive analysis of the resistance mechanisms involved.