Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

Background: New insecticides with novel modes of action such as neonicotinoids have recently been recommended for public health use by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods: Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Atatam), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, acetone+MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.Results: Lower mortalities were observed for all populations when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality for Nkolondom, 31.7- 48.2% for Mangoum, 34.6- 56.1% for Mayuge, 39.4- 45.6% for Atatam, 83.7- 89.3% for Congo and 71.05- 95.9% for Njombe pendja) compared to acetone + MERO for which 100% mortality was observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). Conclusions: This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting in lower mortality, thus possibly overestimating resistance, whereas the addition of MERO consistently increased the efficacy of neonicotinoids in terms of percentage mortalities and time to final mortality. The addition of MERO could however prevent the early detection of resistance development. We therefore recommend monitoring susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Download Full-text

Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

10.21203/rs.3.rs-1197098/v1 ◽

2022 ◽

Author(s):

Magellan Tchouakui ◽

Tatiane Assatse ◽

Leon M. J. Mugenzi ◽

Benjamin D. Menze ◽

Daniel Nguiffo-Nguete ◽

...

Keyword(s):

Democratic Republic Of Congo ◽

Natural Populations ◽

Resistance Mechanisms ◽

Lower Mortality ◽

Malaria Vectors ◽

Cross Resistance ◽

Metabolic Resistance ◽

Resistance Development ◽

Development Of Resistance ◽

Bottle Test

Abstract Background New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.MethodsMosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.ResultsLower mortality were observed when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality in Nkolondom, 31.7- 48.2% in Mangoum, 34.6- 56.1% in Mayµge, 39.4- 45.6% in Obuasi, 83.7- 89.3% in Congo and 71.05- 95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). ConclusionsThis study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Download Full-text

Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, Anopheles funestus

Wellcome Open Research ◽

10.12688/wellcomeopenres.15013.2 ◽

2019 ◽

Vol 4 ◽

pp. 13 ◽

Cited By ~ 1

Author(s):

Magellan Tchouakui ◽

Billy Tene Fossog ◽

Brigitte Vanessa Ngannang ◽

Doumani Djonabaye ◽

Williams Tchapga ◽

...

Keyword(s):

Natural Populations ◽

Anopheles Funestus ◽

Resistance Mechanisms ◽

Malaria Vectors ◽

Glutathione S Transferase ◽

Metabolic Resistance ◽

Mating Competitiveness ◽

Metabolic Marker ◽

Susceptible Allele ◽

Permanet 3.0

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An. funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms. A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.

Download Full-text

Evolution of the Insecticide Target Rdl in African Anopheles Is Driven by Interspecific and Interkaryotypic Introgression

Molecular Biology and Evolution ◽

10.1093/molbev/msaa128 ◽

2020 ◽

Vol 37 (10) ◽

pp. 2900-2917 ◽

Cited By ~ 4

Author(s):

Xavier Grau-Bové ◽

Sean Tomlinson ◽

Andrias O O’Reilly ◽

Nicholas J Harding ◽

Alistair Miles ◽

...

Keyword(s):

Large Scale ◽

Sequence Data ◽

Management Strategies ◽

Natural Populations ◽

Resistance Mechanisms ◽

Health Concern ◽

Malaria Vectors ◽

Resistance Mutations ◽

Development Of Resistance ◽

Evolution Of Resistance

Abstract The evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across nonconcordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors.

Download Full-text

Evolution of the insecticide target Rdl in African Anopheles is driven by interspecific and interkaryotypic introgression

10.1101/2019.12.17.879775 ◽

2019 ◽

Author(s):

Xavier Grau-Bové ◽

Sean Tomlinson ◽

Andrias O. O’Reilly ◽

Nicholas J. Harding ◽

Alistair Miles ◽

...

Keyword(s):

Large Scale ◽

Sequence Data ◽

Management Strategies ◽

Natural Populations ◽

Resistance Mechanisms ◽

Health Concern ◽

Malaria Vectors ◽

Resistance Mutations ◽

Development Of Resistance ◽

Evolution Of Resistance

AbstractThe evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across non-concordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors.

Download Full-text

Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males Anopheles funestus, African malaria vector

Wellcome Open Research ◽

10.12688/wellcomeopenres.15013.1 ◽

2019 ◽

Vol 4 ◽

pp. 13 ◽

Cited By ~ 3

Author(s):

Magellan Tchouakui ◽

Billy Tene Fossog ◽

Brigitte Vanessa Ngannang ◽

Doumani Djonabaye ◽

Williams Tchapga ◽

...

Keyword(s):

Natural Populations ◽

Anopheles Funestus ◽

Resistance Mechanisms ◽

Malaria Vectors ◽

Glutathione S Transferase ◽

Metabolic Resistance ◽

Mating Competitiveness ◽

Metabolic Marker ◽

Susceptible Allele ◽

Permanet 3.0

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An.funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms. A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.

Download Full-text

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽

10.3390/cells9010142 ◽

2020 ◽

Vol 9 (1) ◽

pp. 142 ◽

Cited By ~ 11

Author(s):

Mariusz L. Hartman ◽

Malgorzata Sztiller-Sikorska ◽

Anna Gajos-Michniewicz ◽

Malgorzata Czyz

Keyword(s):

Phenotypic Plasticity ◽

Cell Lines ◽

Mapk Pathway ◽

Acquired Resistance ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Resistant Cell ◽

Cross Resistance ◽

Preclinical Model ◽

Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

Download Full-text

Lactococcus lactis resistance to aureocin A53- and enterocin L50-like bacteriocins and membrane-targeting peptide antibiotics relies on the YsaCB-KinG-LlrG four-component system

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00921-21 ◽

2021 ◽

Author(s):

Aleksandra Tymoszewska ◽

Kirill V. Ovchinnikov ◽

Dzung B. Diep ◽

MaŁgorzata SŁodownik ◽

Edyta Maron ◽

...

Keyword(s):

Lactococcus Lactis ◽

Abc Transporter ◽

Cell Envelope ◽

Resistance Mechanisms ◽

Cross Resistance ◽

Minor Effect ◽

Peptide Antibiotics ◽

Component System ◽

Development Of Resistance ◽

Targeting Peptide

Resistance to non-ribosomally synthesized peptide antibiotics affecting the cell envelope is well-studied and mostly associated with the action of peptide-sensing and detoxification (PSD) modules which consist of a two-component system (TCS) and an ATP-binding cassette (ABC) transporter. In contrast, the resistance mechanisms to ribosomally synthesized bacterial toxic peptides (bacteriocins), which also affect the cell envelope, are studied to lesser extent, and possible cross-resistance between them and antibiotics is still poorly understood. In the present study, we investigated the development of resistance of Lactococcus lactis to aureocin A53- and enterocin L50-like bacteriocins and cross-resistance with antibiotics. First, 19 spontaneous mutants resistant to their representatives were selected and displayed changes in the sensitivity also to peptide antibiotics acting on the cell envelope (bacitracin, daptomycin, and gramicidin). Sequencing of their genomes revealed mutations in genes encoding ABC transporter YsaCB and TCS KinG-LlrG, the emergence of which induced upregulation of the dltABCD and ysaDCB operons. The ysaB mutations were either nonsense or frameshift and led to the generation of truncated YsaB but with the conserved N-terminal FtsX domain intact. Deletions of ysaCB or llrG had a minor effect on the resistance of the obtained mutants to the tested bacteriocins, daptomycin, and gramicidin, indicating that the development of resistance is dependent on the modification of the protein rather than its absence. In further corroboration of the above conclusion, we show that the FtsX domain, which functions effectively when the YsaB is lacking its central and C-terminal parts, is critical for the resistance to these antimicrobials.

Download Full-text

Potential Metabolic Resistance Mechanisms to Ivermectin in Anopheles Gambiae: A Synergist Bioassay Study

10.21203/rs.3.rs-121991/v1 ◽

2020 ◽

Author(s):

Patricia Nicolas ◽

Caroline Kiuru ◽

Martin Wagah ◽

Martha Muturi ◽

Urs Duthaler ◽

...

Keyword(s):

Cytochrome P450 ◽

Anopheles Gambiae ◽

Control Strategies ◽

Resistance Mechanisms ◽

World Health ◽

Malaria Vectors ◽

Malaria Vector Control ◽

Cytochrome P450 Enzymes ◽

Metabolic Resistance ◽

Active Efflux

Abstract Background Despite remarkable success obtained with current malaria vector control strategies in the last 15 years, additional innovative measures will be needed to achieve the ambitious goals set for 2030 by the World Health Organization (WHO). New tools will need to address insecticide resistance and residual transmission as key challenges. Endectocides such as ivermectin are drugs that kill mosquitoes which feed on treated subjects. Mass administration of ivermectin can effectively target outdoor and early biting vectors, complementing the still effective conventional tools. Although this approach has garnered attention, development of ivermectin resistance is a potential pitfall. Herein, we evaluate the potential role of xenobiotic pumps and cytochrome P450 enzymes in protecting mosquitoes against ivermectin by active efflux and metabolic detoxification, respectively. Methods We determined the lethal-concentration 50 for ivermectin in colonized Anopheles gambiae, then we used chemical inhibitors and inducers of xenobiotic pumps and cytochrome P450 enzymes in combination with ivermectin to probe the mechanism of ivermectin detoxification. Results Dual inhibition of xenobiotic pumps and cytochromes have a synergistic effect with ivermectin, greatly increasing mosquito mortality. Inhibition of xenobiotic pumps alone had no effect on ivermectin-induced mortality. Induction of xenobiotic pumps and cytochromes may confer partial protection from ivermectin.Conclusion there is a clear pathway for development of ivermectin resistance in malaria vectors. Detoxification mechanisms mediated by cytochrome P450 enzymes are more important than xenobiotic pumps in protecting mosquitoes against ivermectin.

Download Full-text

Genome-wide gene expression profiling reveals that cuticle alterations and P450 detoxification are associated with pyrethroid resistance in Anopheles arabiensis populations from Ethiopia

10.1101/451336 ◽

2018 ◽

Author(s):

Eba Alemayehu Simma ◽

Wannes Dermauw ◽

Vasileia Balabanidou ◽

Simon Snoeck ◽

Astrid Bryon ◽

...

Keyword(s):

Insecticide Resistance ◽

Anopheles Arabiensis ◽

Pyrethroid Resistance ◽

Resistance Mutation ◽

Resistance Mechanisms ◽

Target Site ◽

Malaria Vectors ◽

Metabolic Resistance ◽

Genome Wide ◽

Target Site Resistance

AbstractBACKGROUNDVector control is the main intervention in malaria control and elimination strategies. However, the development of insecticide resistance is one of the major challenges for controlling malaria vectors. Anopheles arabiensis populations in Ethiopia showed resistance against both DDT and the pyrethroid deltamethrin. Although a L1014F target-site resistance mutation was present in the voltage gated sodium channel of investigated populations, the levels of resistance and biochemical studies indicated the presence of additional resistance mechanisms. In this study, we used genome-wide transcriptome profiling by RNAseq to assess differentially expressed genes between three deltamethrin and DDT resistant An. arabiensis field populations (Tolay, Asendabo, Chewaka) and two susceptible strains (Sekoru and Mozambique).RESULTSBoth RNAseq analysis and RT-qPCR showed that a glutathione-S-transferase, gstd3, and a cytochrome P450 monooxygenase, cyp6p4, were significantly overexpressed in the group of resistant populations compared to the susceptible strains, suggesting that the enzymes they encode play a key role in metabolic resistance against deltamethrin or DDT. Furthermore, a gene ontology enrichment analysis showed that expression changes of cuticle related genes were strongly associated with insecticide resistance, although this did not translate in increased thickness of the procuticle.CONCLUSIONOur transcriptome sequencing of deltamethrin/DDT resistant An. arabiensis populations from Ethiopia suggests non-target site resistance mechanisms and pave the way for further investigation of the role of cuticle composition in resistance.

Download Full-text

Role of metabolic enzymes in resistance to chlorpyrifos-methyl in the cowpea aphid, Aphis craccivora (Koch)

Journal of Plant Protection Research ◽

10.1515/jppr-2017-0039 ◽

2017 ◽

Vol 57 (3) ◽

pp. 275-280

Author(s):

El-Sayed Mohammad Soliman Mokbel ◽

Eman Saed Hassan Swelam ◽

Eman Mohamed Mostafa Radwan ◽

Mohammed Abd-Elhady Kandil

Keyword(s):

Resistance Management ◽

Resistance Mechanisms ◽

Aphis Craccivora ◽

Cross Resistance ◽

Repeated Treatment ◽

Glutathione S Transferase ◽

Metabolic Resistance ◽

Cowpea Aphid ◽

Aphis Craccivora Koch

AbstractThe cowpea aphid,Aphis craccivoramanagement relies mainly on chemical control. As a result extensive and repeated treatment of insecticides has led to the development of aphid resistance to commonly used insecticides. To investigate chlorpyrifos-methyl resistance inA. craccivora, a field strain was selected for 24-generations to achieve a resistance factor of 82.3 fold compared with a susceptible strain. In the resistant strain, malathion and lambda-cyhalothrin exhibited obvious cross-resistance; while fenvalerate and dinotefuran showed moderate cross-resistance. In contrast, slight or no cross-resistance was obtained with the other tested insecticides. To investigate metabolic resistance mechanisms, integration of biochemical and synergism assays was conducted. Results showed the key role of esterase (EST) and mixed function oxidases (MFO); however, glutathione-s-transferase (GST) contributed less to resistance. Cross-resistance studies showed the need for rotation with non-cross resistant insecticides as a resistance management tactic.

Download Full-text