Resistance development,cross-resistance,and fitness costs associated with Aphis gossypii resistance towards sulfoxaflor and acetamiprid in different geographical regions

Aphis gossypii has become increasingly difficult to manage due to its strong insecticide resistance.In the laboratory,we established sulfoxaflor-resistant and acetamiprid-resistant strains in two A.gossypii populations with different basal insecticide resistance levels,and evaluated the effects of basal insecticide resistance on the resistance development and cross-resistance,as well as differences in fitness.Under the same selection pressure,Yarkant A.gossypii(with low basal insecticide resistance)evolved resistance to sulfoxaflor and acetamiprid more quickly than Jinghe A.gossypii(with high basal insecticide resistance),and the evolution of A.gossypii resistance to sulfoxaflor developed faster than acetamiprid in both Yarkant and Jinghe,Xingjiang,China.The sulfoxaflor-resistant strains selected from Yarkant and Jinghe developed significant cross-resistance to acetamiprid,imidacloprid,thiamethoxam and pymetrozine;while the acetamiprid-resistant strains developed significant cross-resistance to sulfoxaflor,imidacloprid,thiamethoxam,pymetrozine,and chlorpyrifos.The relative fitness of A.gossypii decreased as the resistance to sulfoxaflor and acetamiprid developed.The relative fitness levels of the sulfoxaflor-resistant strains(Yarkant-SulR and Jinghe-SulR)were lower than those of the acetamipridresistant strains(Yarkant-AceR and Jinghe-AceR).In addition,the relative fitness levels of sulfoxaflor-and acetamiprid-resistant strains were lower in Jinghe than in Yarkant.In summary,basal insecticide resistance of A.gossypii and insecticide type affected the evolution of resistance to insecticides in A.gossypii,as well as cross-resistance to other insecticides.The sulfoxaflor-and acetamiprid-resistant A.gossypii strains had obvious fitness costs.The results of this work will contribute to the insecticide resistance management and integrated management of A.gossypii.

Cross-resistance pattern to four AHAS-inhibiting herbicides of tribenuron-methyl-resistant flixweed(Descurainia sophia) conferred by Asp-376-Glu mutation in AHAS

Flixweed（Descurainia sophia L.） is a problematic and widespread weed in winter wheat fields and has been controlled by tribenuron-methyl for more than twenty years in China. In this study, a flixweed accession（Hebei 25, HB25） with an Asp-376-Glu mutation in acetohydroxy acid synthase（AHAS） was identified and purified. The purified HB25 accession（p HB25） developed 758.1-fold resistance to tribenuron-methyl and exhibited obvious cross-resistance to four AHAS-inhibiting herbicides. The resistant/susceptible（R/S） ratios of 50% plant growth reduction（GR_（50）） to herbicides of halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 346.1, 15.7, 8.1 and 7.1, respectively. The reduced AHAS sensitivities to four different AHAS-inhibiting herbicides, which were caused by the Asp-376-Glu mutation, were responsible for the resistance and cross-resistance to AHAS-inhibiting herbicides. The R/S ratios of 50% inhibition of AHAS activity（I50） to tribenuron-methyl, halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 844.5, 532.9, 74.5, 13.3 and 5.5, respectively. The results of AHAS activity in vitro were highly correlated with that of whole-plant response experiments.

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis(Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research(including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm(Helicoverpa armigera) to the Bt toxins Cry1 Ac and Cry2 Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1 Ac and Cry2 Ab against cotton bollworm are not completely independent, and then propose the "resistance-associated gene mutation potential hypothesis". Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting "pyramid" strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.

Detection of Resistance and Cross-resistance to Four ALS-inhibiting Herbicides of Scirpus juncoides Roxb. in Paddy Field in Heilongjiang Province

The resistance and cross-resistance of Scirpus juncoides Roxb.to ALS-inhibiting herbicides were tested to provide theoretical basis for effectively control Scirpus juncoides Roxb.and rational use of herbicides.To identify the resistance and crossresistance of 19 populations from nine areas in Heilongjiang Province to four ALS-inhibiting herbicides by agar method and the whole-plant assay.The results are as the followings,the two bioassay methods had good correlation,the resistance indexes in the same area were different by two means,but resistant level was the same,similarity up to 89.5%.The resistant rates to pyrazosulfuronethyl,bensulfuron-methyl,penoxsulam and pyribenzoxim were 26.3%,36.8%,36.1%and 21.1%,respectively.Shangzhi populations evolved the highest level of resistance to pyrazosulfuron-methyl with a resistance index of 15.56.Shangzhi,Tonghe,Fangzheng and Jixi populations showed cross-resistance to four ALS-inhibiting herbicides,accounting for 26.3%.The resistance of Scirpus juncoides Roxb.to four ALS-inhibiting herbicides in Heilongjiang paddy field was low to moderate,five populations showed cross-resistance to four ALS-inhibiting herbicides.

Cross-resistance and genetics of field-evolved resistance to chlorfenapyr in Plutella xylostella

Chlorfenapyr is a broad-spectrum halogenated pyrrole insecticide with a unique mode of action.Due to the misuse and overuse of this chemical,resistance has been reported in several arthropods,including Plutella xylostella,which is one of the most destructive insect pests afflicting crucifers worldwide.A better understanding of the cross-resistance and genetics of field-evolved chlorfenapyr resistance could effectively guide resistance management practices.Here,the chlorfenapyr resistance of a fieldderived population of P xylostella was introgressed into the susceptible IPP-S strain using a selection-assisted multigenerational backcrossing approach.The constructed nearisogenic strain,TH-BCsF2,shared 98.4%genetic background with the recurrent parent IPP-S strain.The TH-BCsF2 strain showed 275-fold resistance to chlorfenapyr,but no significant cross-resistance to spinosad,abamectin,chlorpyrifos,β-cypermethrin,indoxacarb,chlorantraniliprole,or broflanilide(no more than 4.2-fold).Genetic analysis revealed that resistance was autosomal,incompletely dominant,and conferred by 1 major gene or a few tightly linked loci.The synergism of metabolic inhibitors(PBO,DEM,and DEF)to chlorfenapyr was very weak(<1.7-fold),and the metabolic enzyme activities in the TH-BCsF2 strain were not significantly elevated compared with the IPP-S strain.The results enhances our understanding of the genetic traits of chlorfenapyr resistance,and provides essential information for improving resistance management strategies.

Risk assessment,fitness cost,cross-resistance,and mechanism of tetraniliprole resistance in the rice stem borer,Chilo suppressalis

The rice stem borer(RSB),Chilo suppressalis,a notorious rice pest in China,has evolved a high resistance level to commonly used insecticides.Tetraniliprole,a new anthranilic diamide insecticide,effectively controls multiple pests,including RSB.However,the potential resistance risk of RSB to tetraniliprole is still unknown.In this study,the tetraniliprole-selection(Tet-R)strain was obtained through 10 continuous generations of selection with tetraniliprole 30%lethal concentration(LC_(30)).The realized heritability(h^(2))of the Tet-R strain was 0.387,indicating that resistance of RSB to tetraniliprole developed rapidly under the continuous selection of tetraniliprole.The Tet-R strain had a high fitness cost(relative fitness=0.53).We established the susceptibility baseline of RSB to tetraniliprole(lethal concentration at LC_(50)=0.727 mg/L)and investigated the resistance level of 6 field populations to tetraniliprole.All tested strains that had resistance to chlorantraniliprole exhibited moderate-to high-level resistance to tetraniliprole(resistance ratio=27.7-806.8).Detection of ryanodine receptor(RyR)mutations showed that the Y4667C,Y4667D,I4758M,and Y4891F mutations were present in tested RSB field populations.RyR mutations were responsible for the cross-resistance between tetraniliprole and chlorantraniliprole.Further,the clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-mediated genome-modified flies were used to study the contribution of RyR mutations to tetraniliprole resistance.The order of contribution of a single RyR mutation to tetraniliprole resistance was Y4667D>G4915E>Y4667C~~I4758M>Y4891F.In addition,the I4758M and Y4667C double mutations conferred higher tetraniliprole resistance than single Y4667C mutations.These results can guide resistance management practices for diamides in RSB and other arthropods.

The Relationship Between Resistance to Controlled Atmosphere and Insecticides of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The cross resistance of four resistant strains of Liposcelis bostrychophila Badonnel, an important stored-product insect pest, was investigated. These four strains included the HCO2-R (hypercarbia-resistant strain), HCLO-R (hypoxia and hypercarbia resistant strain), DDVP-R (DDVP-resistant strain) and PH3-R (PH3-resistant strain). The results indicated that there were cross-resistances between CA and insecticides, and the quantities of the cross-resistance were different. The cross-resistance factor (RF) of HCO2-R to hypoxia and hypercarbia was 3.2458, whilst, that of HCLO-R to hypercarbia was 1.8280. The RF of DDVP-R to PH3 was 3.9614, whilst, that of PH3-R to DDVP was 2.7852. The RF values of DDVP- R and PH3-R to hypercarbia were 1.3550 and 1.1816, respectively. However, the RF of HCO2- R to DDVP was 2.1372. There also was a low cross-resistance between DDVP-R and HCO2-R. The RF of HCO2-R to PH3 was 3.3698. This suggested that the insects resistant to high CO2 concentration atmosphere would develop significant resistance to PH3. However, the insects resistant to PH3 remain sensitive to hypercarbia atmosphere. Both DDVP-R and PH3- R developed resistance to high CO2 treatment. There was a low cross-resistance between DDVP-R and HCLO-R. The insects resistant to hypoxia and hypercarbia were very sensitive to DDVP. There also was cross-resistance between HCLO-R and PH3-R, but the resistance of PH3-R to hypoxia and hypercarbia was lower than that of HCLO-R to PH3. The difference of the overlapping and separate values indicated that there were differences in the intercross-resistance of four resistant strains.

Characterisation of Neonicotinoid and Pymetrozine Resistance in Strains of Bemisia tabaci(Hemiptera:Aleyrodidae)from China

Four strains of the Q biotype and one of the B biotype of the whitefly Bemisia tabaci collected from China were characterised for resistance to four neonicotinoid insecticides and pymetrozine.Q biotype strains showed moderate to strong resistance to imidacloprid,thiamethoxam and acetamiprid,but little or no cross-resistance to dinotefuron.Resistance to neonicotinoids was consistently associated with resistance to pymetrozine,despite the latter having a distinct（though unresolved）mode of action.The single B biotype strain proved largely susceptible to all the insecticides investigated.Resistance in the Q biotype strains was associated with over-expression of a cytochrome P450 monooxygenase gene,CYP6CM1,whose substrate specificity presumably accounts for the observed cross-resistance profiles.

Characterization of field-evolved resistance to cyantraniliprole in Bemisia tabaci MED from China

Cyantraniliprole is a novel anthranilic diamide insecticide with significant efficacy against Bemisia tabaci,an important pest insect worldwide.In this study,we conducted reversion and selection work and genetic analysis,and determined cross-resistance spectrum and synergism of cyantraniliprole resistance based on the reported population,SX population,of B.tabaci collected from Shanxi Province,China.Compared with a susceptible strain(MED-S),SX population,the field-evolved cyantraniliprole-resistant population exhibited 26.4-fold higher resistance to cyantraniliprole.In SX,a sharp decline of cyantraniliprole resistance was shown in the absence of selection.Another tested strain,SX-R,was established from SX population after successive selection with cyantraniliprole and recently developed 138.4-fold high resistance to cyantraniliprole.SX-R had no cross-resistance to abamectin,imidacloprid,thiamethoxam,sulfoxaflor,or bifenthrin.Genetic analysis illustrated that cyantraniliprole resistance in SX-R was autosomally inherited and incompletely dominant.Additionally,piperonyl butoxide(PBO)significantly inhibited cyantraniliprole resistance in the SX-R strain.In conclusion,the selection of SX with cyantraniliprole led to high resistance to cyantraniliprole which is incompletely dominant and no cross-resistance to several common types of insecticides.Enhanced oxidative metabolism is possibly involved in the resistance of SX-R,yet target-site resistance could not be excluded.

Deltamethrin:Promising mosquito control agent against adult stage of Aedes aegypti L.

Objective:To evaluate the effects of deltamethrin against field-collected adults of Aedes aegypti L(Ae.aegypti).Methods:The adults were selected with 0.025%deltamethrin for 40 successive generations.The selected adults were tested with 4%DDT and the emerging larvae were tested with various insecticides to study the cross-resistance spectrum.The knockdown and irritability studies were carried out in adult mosquitoes to investigate their behavioural response to deltamethrin.Results:Forty generations of selection with deltamethrin resulted in only 3.8-fold resistance in the adults of Ae.aegypti.The adults of parent(PS) and selected strains (DAS) exhibited only 0.8-fold cross resistance to 4%DDT.The larvae emerging from the PS and DAS strains did not develop appreciable levels of resistance to various insecticides tested.The knockdown studies revealed KTSO of 14.4 min in PS adults with no signs of recovery even after 24 h. The DAS strains could develop only 1.2 to 1.3-fold knockdown resistance(KDR).The knockdown response of DDT was though 5-6 times slower than deltamethrin but the continued response in deltamethrin-selected adults caused only 1.2-fold KDR.The PS and DAS strains exhibited significant irritability response towards deltamethrin and DDT.The DAS strains showed 5-6 fold increased irritability to deltamethrin as compared to the PS strain.Conclusions:The above results suggest the prolonged effective use of deltamethrin against Ae.aegypti as an adulticide.

Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass(Beckmannia syzigachne)

American sloughgrass(Beckmannia syzigachne(Steud.) Fernald) is one of the most competitive and malignant weeds in rice-wheat rotation fields in China. American sloughgrass populations in the Jiangsu Province of China became less sensitive to acetohydroxyacid synthase(AHAS) inhibitors after repeated application for many years in these areas. Two suspected resistant American sloughgrass populations(R1 and R2) collected in the field were detected the resistance to inhibitors of AHAS in whole-plant dose-response assays, compared to the susceptible(S) population. These assays indicated that R1 showed low resistance to mesosulfuron-methyl(3.32-fold), imazapic(2.84-fold) and pyroxsulam(1.55-fold), moderate resistance to flazasulfuron(4.67-fold) and pyribenzoxim(7.41-fold), and high resistance to flucarbazone(11.73-fold). However, using a combination of the cytochrome P450 inhibitor, malathion, with mesosulfuron-methyl resulted in a reduction in R1 resistance relative to mesosulfuron-methyl alone. Furthermore, R2 was highly resistant to flazasulfuron(34.90-fold), imazapic(11.30-fold), flucarbazone(49.20-fold), pyribenzoxim(12.94-fold), moderately resistant to mesosulfuron-methyl(9.77-fold) and pyroxsulam(6.26-fold), and malathion had no effect on R2 resistance to mesosulfuron-methyl. The fulllength of AHAS genes was sequenced and the AHAS enzymes were assayed in vitro in order to clarify the mechanism of resistance to AHAS inhibitors in R1 and R2 populations. The results demonstrated that R2 had a Pro-197-Ser mutation in the AHAS gene, and the sensitivity of R2 to the five AHAS inhibitors was decreased, which may result in R2 resistance to AHAS inhibitors. There was no mutation in the AHAS gene of R1, and there were no significant differences in enzyme sensitivity between susceptible(S) and resistant(R1) populations. An enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors.

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera(Hübner)

The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible（96S） and Cry1Ac-resistant H. armigera strain（Cry1Ac-R）. The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance（SPR） showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A＋Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.

<i>gyrA</i>Gene Mutation Conferring Phenotypic Cross-Resistance among Fluoroquinolones (Ofloxacin, Levofloxacin and Gatifloxacin) in Multidrug Resistant <i>Mycobacterium tuberculosis</i>Strains Isolated from Pulmonary MDR-TB Patients in Bangladesh

Background and objectives: Fluoroquinolones (FLQs) are an essential component of multidrug resistant-tuberculosis (MDR-TB) treatment regimen but unfortunately the emergence of FLQ resistant MDR-TB cases is challenging the current MDR-TB treatment regimen. FLQ resistance is mainly caused by gyrA gene mutation and phenotypic cross-resistance may occur among the different FLQs. A limited number of data exists regarding the cross-resistance phenomenon among FLQs and it appears that resistance to the present class representative FLQ, ofloxacin (OFX), may or may not correlate with complete cross-resistance to other FLQs. So the study was designed to observe if gyrA gene mutations confer to the phenotypic cross-resistance among FLQs [OFX, Levofloxacin (LFX) and Gatifloxacin (GFX)] tested. Methodology: Sputum samples from 68 diagnosed pulmonary MDR-TB cases were collected. All samples were subjected to Multiplex Real-time PCR for the detection of gyrA gene mutations and conventional culture on Lowenstein-Jensen (L-J) media followed by drug sensitivity testing (DST) of culture isolates (MDR-TB strains) by indirect proportion method for the detection of phenotypic resistance pattern to OFX, LFX and GFX. Results: Out of the 68 MDR-TB sputum samples 13 (19.11%) had MDR-TB bacilli with mutations in the gyrA gene and 11(16.18%) of the MDR-TB culture isolates were found to have resistance to FLQs by DST. The study observed that 11 MDR-TB samples with gyrA gene mutations showed complete phenotypic cross-resistance among OFX, LFX and GFX. Conclusion: This study found that mutation in the gyrA gene of the MDR-TB bacilli results in the complete cross-resistance among the FLQs (OFX, LFX and GFX) tested. It is therefore of utmost importance to carry out the base line resistance and cross-resistance tests for the individual FLQs prior to initiating the treatment of MDR-TB cases in Bangladesh for successful clinical outcomes.

Failure of Cronobacter sakazakii to acquire direct and cross-protection against lethal stresses after exposure to sublethal ethanol

Cronobacter sakazakii may encounter subinhibitory concentrations of ethanol stress over its lifecycle.Bacterial tolerance to homologous or heterologous stress agents may be altered as a result of ethanol adaptive responses.Therefore,the tolerance of ethanol-exposed and control cells to subsequent lethal stresses was evaluated in the current work.It was discovered that sublethal ethanol exposure increased the susceptibility to lethal ethanol stress in C.sakazakii as determined by the Weibull model.Furthermore,sublethal ethanol concentration exposure in C.sakazakii did not lead to any cross-tolerance against other stressors such as benzalkonium chloride(120 mg/L),heat(55°C),cold(4°C),simulated gastric fuid(pH 3.0),osmotic stress(sorbitol,0.75 g/mL),and desiccation stress.Analysis of zeta potential,scanning electron microscope,and surface-enhanced Raman spectroscopy spectra revealed that cellular injury and changes in cellular chemical composition may contribute to the reduced resistance of C.sakazakii after ethanol exposure.Furthermore,sublethal ethanol exposure resulted in an elevated proportion of unsaturated fatty acids(USFA),while reducing the proportion of saturated fatty acids(SFA)and the ratio of SFA to USFA.The developed inactivation models can serve as a valuable source of data to support quantitative microbial risk assessment.Moreover,a better understanding of the response of C.sakazakii to sublethal ethanol exposure may provide valuable insights into the prevention and control of C.sakazakii.

Resistance and fitness costs in diamondback moths after selection using broflanilide, a novel meta-diamide insecticide

The diamondback moth (DBM) Plutella xylostella (L.) (Lepidoptera: Plutellidae) is an insect pest found around the world that feeds on cruciferous crops. The DBM has become resistant to most insecticides in current use in the field. Broflanilide is a novel meta-diamide insecticide that binds to a new site on the γ-aminobutyric acid receptor and very efficiently protects against most pests in the order Lepidoptera, including DBM. In this study, the resistance of a laboratory-bred susceptible strain of DBM to broflanilide and the fitness costs posed by broflanilide to the DBM were evaluated. The DBM had no obvious resistance to broflanilide after 10 generations of selection. The realized heritability h2 was 0.033, suggesting a low risk of resistance developing in this strain. The F10 generation had no cross-resistance to the insecticides abamectin and endosulfan (which target the γ-aminobutyric acid receptor) and chlorantraniliprole (which targets a non-γ-aminobutyric acid receptor). The specific activities of important detoxification enzymes (cytochrome P450 monooxygenase, esterase, and glutathione S-transferase) were not obviously altered. However, the larval stage was prolonged and the adult stage was shortened significantly in F11 generation than the F0 generation. The total preoviposition period TPOP significantly prolonged 1.90 d in F11 generation. The fitness value Rf (0.93) was lower for the F11 generation than the F0 generation. The results indicated that long-term exposure to broflanilide exerts clear fitness costs in the DBM. This information will be useful in identifying reasonable broflanilide application guidelines for managing broflanilide resistance in the DBM.

From chemical mixtures to antibiotic resistance

In real environment, it is unlikely that contaminants exist singly; environmental contamination with chemical mixtures is a norm. However, the impacts of chemical mixtures on environmental quality and ecosystem health have been overlooked in the past.Among the complex interactions between different contaminants, their relationship with the rise of antibiotic resistance（AR） is an emerging environmental concern. In this paper,we review recent progresses on how chemicals or chemical mixtures promote AR. We propose that, through co-selection, agents causing stress to bacteria may induce AR. The mechanisms for chemical mixtures to promote AR are also discussed. We also propose that,mechanistic understanding of co-selection of chemical mixtures for AR should be a future research priority in environmental health research.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa

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.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(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 againstAnopheles gambiae s.l.In addition,TaqMan assays were used to genotype key pyrethroid-resistant markers inAn.gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin.Results:Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin(11.4-51.9% mortality in Nkolondom,31.7-48.2% in Mangoum,34.6-56.1% in Mayuge,39.4-45.6% in Obuasi,83.7-89.3% in Congo and 71.1-95.9% in Njombe pendja)compared to acetone+MERO for which 100% mortality were observed for all the populations.Similar observations were done for imidacloprid and acetamiprid.Synergist assays(PBO,DEM and DEF)with clothianidin 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 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(4μg/ml for clothianidin)to capture the accurate resistance profile of the mosquito populations.