Anopheles gambiae larvae's ability to grow and emerge in water containing lethal concentrations of clothianidin, acetamiprid, or imidacloprid is consistent with cross-resistance to neonicotinoids.

BACKGROUND: For decades, various agrochemicals have been successfully repurposed for mosquito control. However, preexisting resistance caused in larval and adult populations by unintentional pesticide exposure or other cross-resistance mechanisms poses a challenge to the efficacy of this strategy. A better understanding of larval adaptation to the lethal and sublethal effects of residual pesticides in aquatic habitats would provide vital information for assessing the efficacy of repurposed agrochemicals against mosquitoes. METHODS: We reared field-collected mosquito larvae in water containing a concentration of agrochemical causing 100% mortality in susceptible mosquitoes after 24 h (lethal concentration). Using this experimental setup, we tested the effect of lethal concentrations of a pyrrole (chlorfenapyr, 0.10 mg/l), a pyrethroid (deltamethrin, 1.5 mg/l), and three neonicotinoids including imidacloprid (0.075 mg/l), acetamiprid (0.15 mg/l), and clothianidin (0.035 mg/l) on mortality rates, growth, and survival in third-instar larvae of the two sibling species Anopheles gambiae and Anopheles coluzzii collected from Yaoundé, Cameroon. RESULTS: We found that An. gambiae and An. coluzzii larvae were susceptible to chlorfenapyr and were killed within 24 h by a nominal concentration of 0.10 mg/l. Consistent with strong resistance, deltamethrin induced low mortality in both species. Lethal concentrations of acetamiprid, imidacloprid, and clothianidin strongly inhibited survival, growth, and emergence in An. coluzzii larvae. By contrast, depending on the active ingredient and the population tested, 5-60% of immature stages of An. gambiae were able to grow and emerge in water containing a lethal concentration of neonicotinoids, suggesting cross-resistance to this class of insecticides. CONCLUSIONS: These findings corroborate susceptibility profiles observed in adults and suggest that unintentional pesticide exposure or other cross-resistance processes could contribute to the development of resistance to neonicotinoids in some Anopheles populations.

Adult mosquitoes of the sibling species Anopheles gambiae and Anopheles coluzzii exhibit contrasting patterns of susceptibility to four neonicotinoid insecticides along an urban-to-rural gradient in Yaoundé, Cameroon.

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.

Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying.

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.

Vegetable oil-based surfactants are adjuvants that enhance the efficacy of neonicotinoid insecticides and can bias susceptibility testing in adult mosquitoes.

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.

Vegetable oil surfactants are synergists that can bias neonicotinoid susceptibility testing in adult mosquitoes.

Background: The standard operating procedure for testing the susceptibility of adult mosquitoes to clothianidin, a neonicotinoid, recommends using a vegetable oil ester as surfactant. However, it has not yet been determined if the surfactant is an inert ingredient or if it can act as a synergist and bias the test. Methodology/Principal Findings: Using standard bioassays, we tested the synergistic effects of a vegetable oil surfactant on a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). Three different formulations of linseed oil soap used as surfactant were far more effective than the standard insecticide synergist piperonyl butoxide in enhancing neonicotinoid activity in Anopheles mosquitoes. At the concentration used in the standard operating procedure (1% v/v), vegetable oil surfactants lead to more than 10-fold reduction in lethal concentrations, LC 50 and LC 99 , of clothianidin in a multi-resistant field population and in a susceptible strain of Anopheles gambiae . At 1% or 0.5% (v/v), the surfactant restored susceptibility to clothianidin, thiamethoxam and imidacloprid and increased mortality to acetamiprid from 43 ± 5.63% to 89 ± 3.25% (P<0.05) in resistant mosquitoes. By contrast, linseed oil soap had no effect on the level of resistance to permethrin and deltamethrin suggesting that the synergism of vegetable oil surfactants may be specific to neoniconoids. Conclusions/Significance: Our findings indicate that vegetable oil surfactants are not inert ingredients in neonicotinoid formulations, and their synergistic effects undermine the ability of standard testing procedures to detect early stages of resistance.

Enhancing the efficacy of neonicotinoids against mosquitoes and overcoming resistance issues.

Background: Neonicotinoids are potential alternatives for targeting pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of Sub-Saharan Africa has yet to be investigated. Here we tested and compared the efficacy of four neonicotinoids alone or in combination with a synergist against two major vectors of Plasmodium . Results: Using standard bioassays, we first assessed the lethal toxicity of three active ingredients against adults of two susceptible Anopheles strains and we determined discriminating doses for monitoring susceptibility in wild populations. We then tested the susceptibility of 5532 Anopheles mosquitoes collected from urban and rural areas of Yaoundé, Cameroon, to discriminating doses of acetamiprid, imidacloprid, clothianidin and thiamethoxam. We found that in comparison with some public health insecticides, neonicotinoids have high lethal concentration, LC 99 , reflecting their low toxicity to Anopheles mosquitoes. In addition to this reduced toxicity, resistance to the four neonicotinoids tested was detected in An. gambiae populations collected from agricultural areas where larvae are intensively exposed to crop-protection neonicotinoids. However, adults of another major vector that occurred in urbanized settings, An. coluzzii , were fully susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. Importantly, the cytochrome inhibitor, piperonyl butoxide (PBO), was very effective in enhancing the activity of clothianidin and acetamiprid providing opportunities to create potent neonicotinoid formulations against Anopheles . Conclusion: These findings suggest that to successfully repurpose agricultural neonicotinoids for malaria vector control, it is essential to use formulations containing synergists such as PBO or surfactants to ensure optimal efficacy.

Chronic exposure of mosquito larvae to pesticide residues endangers a new generation of agrochemicals repurposed for malaria prevention.

Agrochemicals have been successfully repurposed to control mosquitoes worldwide, but pesticides used in agriculture challenge their effectiveness by contaminating surface waters and helping larval populations develop resistance. Thus, knowledge of the lethal and sublethal effects of residual pesticide exposure on mosquitoes is critical for selecting effective insecticides. Here we implemented a new experimental approach to predict the efficacy of agricultural pesticides newly repurposed for malaria vector control. We mimicked insecticide resistance selection as it occurs in contaminated aquatic habitats by rearing field-collected mosquito larvae in water containing a dose of insecticide capable of killing individuals from a susceptible strain within 24 h. We then simultaneously monitored short-term lethal toxicity within 24 h and sublethal effects for 7 days. We found that due to chronic exposure to agricultural pesticides, some mosquito populations are currently pre-adapt to resist neonicotinoids if those were used in vector control. Larvae collected from rural and agricultural areas where neonicotinoid formulations are intensively used for insect pest management were able to survive, grow, pupate and emerge in water containing a lethal dose of acetamiprid, imidacloprid or clothianidin. These results emphasize the importance of addressing exposure of larval populations to formulations applied in agriculture prior to using agrochemicals against malaria vectors.

Resistance to clothianidin reduces the efficacy of SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying, against Anopheles gambiae.

Chronic exposure of mosquito larvae to pesticide residues in agricultural areas is often associated with evolution of resistance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid qualified for Indoor Residual Spraying (IRS). Using standard bioassays, we tested if reduced susceptibility to clothianidin affects the efficacy of SumiShield® 50WG, one of the two newly approved formulations, which contains 50% clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield® 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp collected from urban, suburban and agricultural areas of Yaoundé. We found that the level of susceptibility to the active ingredient predicted the efficacy of SumiShield® 50WG. This formulation was very potent against populations that achieved 100% mortality within 72 h of exposure to a discriminating dose 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 spraying of acetamiprid and imidacloprid is driving cross-resistance to clothianidin. These findings indicate that more potent formulations of clothianidin or different insecticides should be prioritized in areas where resistance is emerging.

Antistaphylococcal Activity of Extracts, Fractions, and Compounds of Acacia polyacantha Wild (Fabaceae).

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), ß-amyrin (2), 3-O-methyl-D-chiro-inositol (3), epicatechin (4), quercetin-3-O-galactoside (5), 3-O-[ß-D-xylopyranosyl-(1 ⟶ 4)-ß-D-galactopyranosyl]-oleanolic acid (6), 3-O-[ß-galactopyranosyl-(1⟶ 4)-ß-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 µg/mL. Fractions exhibited better activities compared to those of their extracts of origin, as their MICs ranged from 16 to 512 µg/mL, with fractions from leaves being more active than those obtained from barks. Compounds had varying activities; MICs varied from 16 to 512 µg/mL with compound 4 presenting the best activity as MICs ≤100 µg/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.

Antibacterial and Antibiotic Modifying Potential of Crude Extracts, Fractions, and Compounds from Acacia polyacantha Willd. against MDR Gram-Negative Bacteria.

The present study aimed to assess the in vitro antibacterial and antibiotic modifying activities of methanol extracts prepared from the leaf (APL) and bark (APB) of Acacia polyacantha, fractions (APLa-d) and compounds isolated from APL against a panel of multidrug resistant (MDR) Gram-negative bacteria. Leaf extract was subjected to column chromatography for compounds isolation; antibacterial assays were performed on samples alone and with an efflux pump inhibitor (EPI), respectively, and several antibiotics on the tested bacteria. The phytochemical investigation of APL led to the isolation of stigmasterol (1), ß-amyrin (2), 3-O-ß-D-glucopyranosylstigmasterol (3), 3-O-methyl-D-chiro-inositol (4), epicatechin (5), quercetin-3-O-glucoside (6), 3-O-[ß-D-xylopyranosyl-(1→4)-ß-D-galactopyranosyl]-oleanolic acid (7), and 3-O-[ß-galactopyranosyl-(1→4)-ß-D-galactopyranosyl]-oleanolic acid (8). APL and APB had minimal inhibitory concentration (MIC) values ≤ 1024 µg/mL on 73.3% and 46.7% of the tested bacteria, respectively. APLb and APLd were effective against 88.9% of tested bacterial species with compound 8 showing the highest activity inhibiting 88.9% of tested bacteria. The EPI, phenylalanine-arginine-ß-naphthylamide (PAßN), strongly improved the activity of APL, APLb, APLd, and compound 8 on all tested bacteria. Synergistic effects were obtained when APL and compounds 7 and 8 were combined with erythromycin (ERY), gentamycin (GEN), ciprofloxacin (CIP), and norfloxacin (NOR). The present study demonstrates the antibacterial potential of Acacia polyacantha and its constituents to combat bacterial infections alone or in combination with EPI.