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

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.

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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.

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An Experimental Hut Evaluation of PBO-Based and Pyrethroid-Only Nets against the Malaria Vector Anopheles funestus Reveals a Loss of Bed Nets Efficacy Associated with GSTe2 Metabolic Resistance

Genes ◽

10.3390/genes11020143 ◽

2020 ◽

Vol 11 (2) ◽

pp. 143 ◽

Cited By ~ 5

Author(s):

Benjamin D. Menze ◽

Mersimine F. Kouamo ◽

Murielle J. Wondji ◽

Williams Tchapga ◽

Micareme Tchoupo ◽

...

Keyword(s):

Anopheles Funestus ◽

Blood Feeding ◽

Malaria Vectors ◽

Bed Nets ◽

Glutathione S Transferase ◽

Metabolic Resistance ◽

Experimental Hut ◽

The Impact ◽

Long Lasting Insecticidal Nets ◽

Permanet 3.0

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

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Fitness Costs of the Glutathione S-Transferase Epsilon 2 (L119F-GSTe2) Mediated Metabolic Resistance to Insecticides in the Major African Malaria Vector Anopheles Funestus

Genes ◽

10.3390/genes9120645 ◽

2018 ◽

Vol 9 (12) ◽

pp. 645 ◽

Cited By ~ 19

Author(s):

Magellan Tchouakui ◽

Jacob M. Riveron ◽

Doumani Djonabaye ◽

Williams Tchapga ◽

Helen Irving ◽

...

Keyword(s):

Management Strategies ◽

Resistance Management ◽

Anopheles Funestus ◽

Adult Emergence ◽

Malaria Vectors ◽

Adult Longevity ◽

Heterozygote Advantage ◽

Glutathione S Transferase ◽

Metabolic Resistance ◽

Life Traits

Metabolic resistance to insecticides threatens malaria control. However, little is known about its fitness cost in field populations of malaria vectors, thus limiting the design of suitable resistance management strategies. Here, we assessed the association between the glutathione S-transferase GSTe2-mediated metabolic resistance and life-traits of natural populations of Anopheles funestus. A total of 1200 indoor resting blood-fed female An. funestus (F0) were collected in Mibellon, Cameroon (2016/2017), and allowed to lay eggs individually. Genotyping of F1 mosquitoes for the L119F-GSTE2 mutation revealed that L/L119-homozygote susceptible (SS) mosquitoes significantly laid more eggs than heterozygotes L119F-RS (odds ratio (OR) = 2.06; p < 0.0001) and homozygote resistant 119F/F-RR (OR = 2.93; p < 0.0001). L/L119-SS susceptible mosquitoes also showed the higher ability for oviposition than 119F/F-RR resistant (OR = 2.68; p = 0.0002) indicating a reduced fecundity in resistant mosquitoes. Furthermore, L119F-RS larvae developed faster (nine days) than L119F-RR and L119F-SS (11 days) (X2 = 11.052; degree of freedom (df) = 4; p = 0.02) suggesting a heterozygote advantage effect for larval development. Interestingly, L/L119-SS developed faster than 119F/F-RR (OR = 5.3; p < 0.0001) revealing an increased developmental time in resistant mosquitoes. However, genotyping and sequencing revealed that L119F-RR mosquitoes exhibited a higher adult longevity compared to RS (OR > 2.2; p < 0.05) and SS (OR > 2.1; p < 0.05) with an increased frequency of GSTe2-resistant haplotypes in mosquitoes of D30 after adult emergence. Additionally, comparison of the expression of GSTe2 revealed a significantly increased expression from D1-D30 after emergence of adults (Anova test (F) = 8; df= 3; p = 0.008). The negative association between GSTe2 and some life traits of An. funestus could facilitate new resistance management strategies. However, the increased longevity of GSTe2-resistant mosquitoes suggests that an increase in resistance could exacerbate malaria transmission.

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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.

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

10.20944/preprints202201.0023.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 ◽

Malaria Vectors ◽

Cross Resistance ◽

Metabolic Resistance ◽

Development Of Resistance ◽

Bottle Test ◽

L1014f Kdr ◽

Anopheles Gambiae S.L

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.

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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.

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Cytochrome P450 metabolic resistance (CYP6P9a) to pyrethroids imposes a fitness cost in the major African malaria vector Anopheles funestus

Heredity ◽

10.1038/s41437-020-0304-1 ◽

2020 ◽

Vol 124 (5) ◽

pp. 621-632 ◽

Cited By ~ 3

Author(s):

Magellan Tchouakui ◽

Jacob Riveron Miranda ◽

Leon M. J. Mugenzi ◽

Doumani Djonabaye ◽

Murielle J. Wondji ◽

...

Keyword(s):

Malaria Vector ◽

Management Strategy ◽

Management Strategies ◽

Resistance Management ◽

Anopheles Funestus ◽

Fitness Cost ◽

Malaria Vectors ◽

Hybrid Strain ◽

Metabolic Resistance ◽

The Impact

Abstract Metabolic resistance threatens the sustainability of pyrethroid-based malaria control interventions. Elucidating the fitness cost and potential reversal of metabolic resistance is crucial to design suitable resistance management strategies. Here, we deciphered the fitness cost associated with the CYP6P9a (P450-mediated metabolic resistance) in the major African malaria vector Anopheles funestus. Reciprocal crosses were performed between a pyrethroid susceptible (FANG) and resistant (FUMOZ-R) laboratory strains and the hybrid strains showed intermediate resistance. Genotyping the CYP6P9a-R resistance allele in oviposited females revealed that CYP6P9a negatively impacts the fecundity as homozygote susceptible mosquitoes (CYP6P9a-SS) lay more eggs than heterozygote (OR = 2.04: P = 0.01) and homozygote resistant mosquitoes. CYP6P9a also imposes a significant fitness cost on the larval development as homozygote resistant larvae (CYP6P9a-RR) developed significantly slower than heterozygote and homozygote susceptible mosquitoes (χ2 = 11.2; P = 0.0008). This fitness cost was further supported by the late pupation of homozygote resistant than susceptible mosquitoes (OR = 2.50; P < 0.01). However, CYP6P9a does not impact the longevity as no difference was observed in the life span of mosquitoes with different genotypes (χ2 = 1.6; P = 0.9). In this hybrid strain, a significant decrease of the resistant CYP6P9a-RR genotype was observed after ten generations (χ2 = 6.6; P = 0.01) suggesting a reversal of P450-based resistance in the absence of selection. This study shows that the P450-mediated metabolic resistance imposes a high fitness cost in malaria vectors supporting that a resistance management strategy based on rotation could help mitigate the impact of such resistance.

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Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms

PLoS ONE ◽

10.1371/journal.pone.0110058 ◽

2014 ◽

Vol 9 (10) ◽

pp. e110058 ◽

Cited By ~ 67

Author(s):

Charles Mulamba ◽

Jacob M. Riveron ◽

Sulaiman S. Ibrahim ◽

Helen Irving ◽

Kayla G. Barnes ◽

...

Keyword(s):

East Africa ◽

Malaria Vector ◽

Anopheles Funestus ◽

Resistance Mechanisms ◽

Metabolic Resistance ◽

Ddt Resistance ◽

Major Malaria Vector

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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.

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CYP6P9-Driven Signatures of Selective Sweep of Metabolic Resistance to Pyrethroids in the Malaria Vector Anopheles funestus Reveal Contemporary Barriers to Gene Flow

Genes ◽

10.3390/genes11111314 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1314

Author(s):

Delia Doreen Djuicy ◽

Jack Hearn ◽

Magellan Tchouakui ◽

Murielle J. Wondji ◽

Helen Irving ◽

...

Keyword(s):

Gene Flow ◽

Selective Sweep ◽

Pyrethroid Resistance ◽

Resistance Mechanism ◽

Anopheles Funestus ◽

Central Africa ◽

Eastern Africa ◽

Malaria Vectors ◽

Metabolic Resistance ◽

Diversity Pattern

Pyrethroid resistance in major malaria vectors such as Anopheles funestus threatens malaria control efforts in Africa. Cytochrome P450-mediated metabolic resistance is best understood for CYP6P9 genes in southern Africa in An. funestus. However, we do not know if this resistance mechanism is spreading across Africa and how it relates to broader patterns of gene flow across the continent. Nucleotide diversity of the CYP6P9a gene and the diversity pattern of five gene fragments spanning a region of 120 kb around the CYP6P9a gene were surveyed in mosquitoes from southern, eastern and central Africa. These analyses revealed that a Cyp6P9a resistance-associated allele has swept through southern and eastern Africa and is now fixed in these regions. A similar diversity profile was observed when analysing genomic regions located 34 kb upstream to 86 kb downstream of the CYP6P9a locus, concordant with a selective sweep throughout the rp1 locus. We identify reduced gene flow between southern/eastern Africa and central Africa, which we hypothesise is due to the Great Rift Valley. These potential barriers to gene flow are likely to prevent or slow the spread of CYP6P9-based resistance mechanism to other parts of Africa and would to be considered in future vector control interventions such as gene drive.

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