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New insecticides prequalified for malaria control interventions include modulators of nicotinic acetylcholine receptors that act selectively on different subunits leading to variable sensitivity among arthropods. This study aimed to investigate the molecular mechanisms underlying contrasting susceptibility to neonicotinoids observed in wild populations of two mosquito sibling species. Bioassays and a synergist test with piperonyl butoxide revealed that the sister taxa, Anopheles gambiae and An. coluzzii, from Yaounde, Cameroon, both have the potential to develop resistance to acetamiprid through cytochrome P450-mediated detoxification. However, contrary to An. coluzzii, An. gambiae populations are evolving cross-resistance to several active ingredients facilitated by mutations of nicotinic acetylcholine receptors (nAChRs). We sequenced coding regions on the β1 and α6 nAChR subunits where variants associated with resistance to neonicotinoids or to spinosyns have been found in agricultural pests and detected no mutation in An. coluzzii. By contrast, six nucleotide substitutions including an amino acid change in one of the loops that modulate ligand binding and affect sensitivity were present in the resistant species, An. gambiae. Allele frequency distributions were consistent with the spread of beneficial mutations that likely reduce the affinity of An. gambiae nAChRs for synthetic modulators. Our findings provide critical information for the application and resistance management of nAChR modulators in malaria prevention., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Neonicotinoid insecticides act selectively on their nicotinic receptor targets leading to variable sensitivity among arthropods. This study aimed to investigate the molecular mechanisms underlying contrasting susceptibility to neonicotinoids observed in wild populations of two mosquito sibling species. Bioassays and a synergism test revealed that the sister taxa, Anopheles gambiae and An. coluzzii , from Yaounde, Cameroon, rely on cytochrome P450s to detoxify neonicotinoids and develop resistance. However, contrary to An. coluzzii , An. gambiae populations are evolving stronger resistance to several active ingredients facilitated by mutations and reduced expression of nicotinic acetylcholine receptors. Six mutations were detected in coding sequences of the β1 and α6 subunits, including two substitutions in one of the loops that modulate ligand binding and sensitivity. Allele frequencies were strongly correlated with a susceptibility gradient between An. coluzzii and An. gambiae suggesting that the mutations may play a key role in sensitivity. Messenger RNA expression levels of the β1, α3, and α7 subunits decreased dramatically, on average by 23.27, 17.50, 15.80-fold, respectively, in wild An. gambiae populations compared to a susceptible insectary colony. By contrast, only the β2 and α9-1 subunits were moderately downregulated (5.28 and 2.67-fold change, respectively) in field-collected An. coluzzii adults relative to susceptible colonized mosquitoes. Our findings provide critical information for the application and resistance management of neonicotinoids in malaria prevention.

Background: Neonicotinoids are potential alternatives for controlling pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of sub-Saharan Africa has yet to be investigated. The aim of the present study was to test the efficacy of four neonicotinoids against adult populations of the sibling species Anopheles gambiae and Anopheles coluzzii sampled along an urban-to-rural gradient., Methods: The lethal toxicity of three active ingredients for adults of two susceptible Anopheles strains was assessed using concentration-response assays, and their discriminating concentrations were calculated. The discriminating concentrations were then used to test the susceptibility of An. gambiae and An. coluzzii mosquitoes collected from urban, suburban and rural areas of Yaoundé, Cameroon, to acetamiprid, imidacloprid, clothianidin and thiamethoxam., Results: Lethal concentrations of neonicotinoids were relatively high suggesting that this class of insecticides has low toxicity against Anopheles mosquitoes. Reduced susceptibility to the four neonicotinoids tested was detected in An. gambiae populations collected from rural and suburban areas. By contrast, adults of An. coluzzii that occurred in urbanized settings were susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. The cytochrome inhibitor, piperonyl butoxide (PBO), significantly enhanced the activity of clothianidin and acetamiprid against An. gambiae mosquitoes., Conclusions: These findings corroborate susceptibility profiles observed in larvae and highlight a significant variation in tolerance to neonicotinoids between An. gambiae and An. coluzzii populations from Yaoundé. Further studies are needed to disentangle the role of exposure to agricultural pesticides and of cross-resistance mechanisms in the development of neonicotinoid resistance in some Anopheles species., (© 2024. The Author(s).)

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

Background: The standard operating procedure for testing the susceptibility of adult mosquitoes to neonicotinoid or butenolide insecticides recommends using a vegetable oil ester (Mero) as a surfactant. However, there is growing evidence that this adjuvant contains surfactants that can enhance insecticide activity, mask resistance and bias the bioassay., Methodology/principal Findings: Using standard bioassays, we tested the effects of commercial formulations of vegetable oil-based surfactants similar to Mero on the activity of a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). We found that three different brands of linseed oil soap used as cleaning products drastically enhanced neonicotinoid activity in Anopheles mosquitoes. At 1% (v/v), the surfactant reduced the median lethal concentration, LC50, of clothianidin more than 10-fold both in susceptible and in resistant populations of Anopheles gambiae. At 1% or 0.5% (v/v), linseed oil soap restored the susceptibility of adult mosquitoes fully to clothianidin, thiamethoxam and imidacloprid and partially to acetamiprid. By contrast, adding soap to the active ingredient did not significantly affect the level of resistance to permethrin or deltamethrin suggesting that vegetable oil-based surfactants specifically enhance the potency of some classes of insecticides., Conclusions/significance: Our findings indicate that surfactants are not inert ingredients, and their use in susceptibility testing may jeopardize the ability to detect resistance. Further research is needed to evaluate the potential, the limitations and the challenges of using some surfactants as adjuvants to enhance the potency of some chemicals applied in mosquito control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ashu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acacia polyacantha is a medicinal plant traditionally used to treat livestock diseases and gastrointestinal infections; our study was undertaken to evaluate the antistaphylococcal activities of the methanolic leaf, bark, and root extracts, fractions, and compounds from Acacia polyacantha against a panel of 14 multidrug-resistant Staphylococcus bacterial strains overexpressing efflux pumps. The study was also extended to investigate two possible modes of action, that is, influence on bacterial growth kinetics and influence on proton-ATPase pumps, of the most active compound against a reference strain. Materials and Methods . The crude extracts after extraction were subjected to column chromatography. Antibacterial assays of extracts, fractions, and compounds alone and in the presence of efflux pump inhibitors were carried out using the broth microdilution method and the study of two mechanisms of action achieved by standard methods with the most active compound. Results . The phytochemical study of Acacia polyacantha leaves leads to the isolation of stigmasterol ( 1 ), β -amyrin ( 2 ), 3- O -methyl- D -chiro-inositol ( 3 ), epicatechin ( 4 ), quercetin-3- O -galactoside ( 5 ), 3- O -[ β - D -xylopyranosyl-(1 ⟶ 4)- β - D -galactopyranosyl]-oleanolic acid ( 6 ), 3- O -[ β -galactopyranosyl-(1⟶ 4)- β - D -galactopyranosyl]-oleanolic acid ( 7 ) and that of leaves lead to the isolation of lupeol ( 8 ) 2,3-dihydroxypropyltetracosanoate ( 9), and methyl-gallate ( 10 ). Leaf, root, and bark extracts inhibited 92.85% (13/14), 92.85% (13/14), and 71.43 % (10/14) of the tested bacteria strains, respectively, with minimum inhibitory concentration (MIC) varying between 16 and 1024  μ g/mL. Fractions exhibited better activities compared to those of their extracts of origin, as their MICs ranged from 16 to 512  μ g/mL, with fractions from leaves being more active than those obtained from barks. Compounds had varying activities; MICs varied from 16 to 512  μ g/mL with compound 4 presenting the best activity as MICs ≤100  μ g/mL were obtained against 11 of the tested bacteria. The activities of extracts, fractions, and compounds were improved in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) as an efflux pump inhibitor to as much as >128 folds. Meanwhile, in the presence of chlorpromazine as an efflux pump inhibitor, only the activity of compound 10 was improved on 10 of the tested bacteria strains. Compound 4 prolonged the lag phase of the growth kinetic in a concentration-dependent manner and equally inhibited the proton-ATPase pumps of the tested bacteria strains. Conclusion . The present study demonstrates the antistaphylococcal potential of Acacia polyacantha and its constituents to combat bacterial infections alone or in combination with efflux pump inhibitors., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Fred A. Ashu et al.)