Variability in resistance-related enzyme activities in field populations of the tarnished plant bug, Lygus lineolaris

Widespread use of Bt crops for control of lepidopterous pests has reduced insecticide use and provided the tarnished plant bug the opportunity to become a serious pest on mid-South cotton. Organophosphate insecticides have predominantly been used against plant bugs in recent years due to the reduced efficacy of other insecticides. In this study, a biochemical approach was developed to survey enzymatic levels associated with organophosphate resistance levels in field populations of the tarnished plant bug. Forty-three populations were collected from the delta areas of Arkansas, Louisiana, and Mississippi. Three esterase substrates and one substrate each of glutathione S-transferase (GST) and acetylcholinesterase (AChE) were used to determine corresponding detoxification enzyme activities in different populations. Compared to a laboratory susceptible colony, increases up to 5.29-fold for esterase, 1.96-fold for GST, and 1.97-fold for AChE activities were detected in the field populations. In addition to the survey of enzyme activities among the populations, we also examined the susceptibility of major detoxification enzymes to several inhibitors which could be used in formulations to synergize insecticide toxicity against the target pests. As much as 52-76% of esterase, 72-98% of GST, and 93% of AChE activities were inhibited in vitro. Revealing variable esterase and GST activities among field populations may lead to a better understanding of resistance mechanisms in the tarnished plant bug. This study also reports effective suppression of detoxification enzymes which may be useful in future insecticide resistance management program for the tarnished plant bug and other Heteropteran pests on Bt crops.     

Variation of acephate susceptibility and correlation with esterase and glutathione S-transferase activities in field populations of the tarnished plant bug, Lygus lineolaris

The tarnished plant bug (TPB) has increasingly become an economically important pest of cotton. Heavy dependence on insecticides, particularly organophosphates and pyrethroids, for TPB control facilitated resistance development to multiple classes of insecticides. To better understand resistance and explore ways to monitor resistance in field populations, this study examined acephate susceptibility and the activities of two major detoxification enzymes in nine field populations collected in the Delta region of Mississippi and Arkansas in 2010. Two Arkansas populations from Reed and Backgate had 3.5- and 4.3-fold resistance to acephate, as compared to a susceptible laboratory strain. Extensive planting of cotton and heavy chemical sprays is a major driving force for resistance development to acephate in Mid-south cotton growing areas. Reduced susceptibility to acephate was highly correlated with elevated esterase activities. The acephate-resistant populations from Backgate, Lula, and Reed consistently had higher (up to 5.3-fold) esterase activities than susceptible populations. Regression analysis of LC₅₀s with kinetic esterase activities revealed a significant polynomial quadratic relationship with R² up to 0.89. Glutathione S-transferase (GST) also had elevated activity in most populations, but the variations of GST activities were not significantly correlated with changes of acephate susceptibility. Finally, examination of esterase and GST inhibitors indicated that suppression rates (up to 70%) by two esterase inhibitors in 2010 were slightly lower than those detected in 2006, and ethacrynic acid (EA) inhibited GST effectively in both years. Two other GST inhibitors (sulfobromophthalein and diethyl maleate) displayed significantly lower suppression rates in 2010 than those detected in 2006, suggesting a potential genetic shift in pest populations and a necessity of continued monitoring for insecticide resistance with both bioassay and biochemical approaches. Results indicated that using major detoxification enzyme activities for resistance monitoring may provide insight into acephate resistance in field populations of TPB.     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

Characterization of acephate resistance in the diamondback moth Plutella xylostella

Decreased acetylcholinesterase (AChE) sensitivity and metabolic detoxification mediated by glutathione S-transferases (GSTs) were examined for their involvement in resistance to acephate in the diamondback moth, Plutella xylostella. The resistant strain showed 47.5-fold higher acephate resistance than the susceptible strain had. However, the resistant strain was only 2.3-fold more resistant to prothiofos than the susceptible strain. The resistant strain included insects having the A298S and G324A mutations in AChE1, which are reportedly involved in prothiofos resistance in P. xylostella, showing reduced AChE sensitivity to inhibition by methamidophos, suggesting that decreased AChE1 sensitivity is one factor conferring acephate resistance. However, allele frequencies at both mutation sites in the resistant strain were low (only 26%). These results suggest that other factors such as GSTs are involved in acephate resistance. Expression of GST genes available in P. xylostella to date was examined using the resistant and susceptible strains, revealing no significant correlation between the expression and resistance levels.     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Resistance mechanisms and associated mutations in acetylcholinesterase genes in Sitobion avenae (Fabricius)

Wheat aphid, Sitobion avenae (fabricius), is one of the most important wheat pests and has been reported to be resistant to commonly used insecticides in China. To determine the resistance mechanism, the resistant and susceptible strains were developed in laboratory and comparably studied. A bioassay revealed that the resistant strain showed high resistance to pirimicarb (RR: 161.8), moderate reistance to omethoate (32.5) and monocrotophos (33.5), and low resistance to deltamethrin (6.3) and thiodicarb (5.5). A biochemistry analysis showed that both strains had similar glutathione-S-transferase (GST) activity, but the resistant strain had 3.8-fold higher esterase activity, and its AChE was insensitive to this treatment. The I₅₀ increased by 25.8-, 10.7-, and 10.4-folds for pirimicarb, omethoate, and monocrotophos, respectively, demonstrating that GST had not been involved in the resistance of S. avenae. The enhanced esterase contributed to low level resistance to all the insecticides tested, whereas higher resistance to pirimicarb, omethoate, and monocrotophos mainly depended on AChE insensitivity. However, the AChE of the resistant strain was still sensitive to thiodicarb (1.7-fold). Thus, thiodicarb could be used as substitute for control of the resistant S. avenae in this case. Furthermore, the two different AChE genes cloned from different resistant and susceptible individuals were also compared. Two mutations, L436(336)S in Sa.Ace1 and W516(435)R in Sa.Ace2, were found consistently associated with the insensitivity of AChE. They were thought to be the possible resistance mutations, but further work is needed to confirm this hypothesis.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

Pyrethroid synergists suppress esterase-mediated resistance in Indian strains of the cotton bollworm, Helicoverpa armigera (Hübner)

The role of esterase in pyrethroid resistance was studied in the final larval instar of different strains of the cotton bollworm, Helicoverpa armigera. The resistant strains viz., Nagpur strain and the Delhi strain were found to have elevated midgut esterase activity in comparison to the susceptible strain. Nagpur strain and Delhi strain have 2.24 and 1.73-fold higher esterase activity, respectively, than that of the susceptible strain. The Native PAGE displayed important differences in the midgut esterase isozyme pattern between the susceptible and the pyrethroid-resistant strains. Out of the 10 esterase isozyme observed, susceptible strain lacked three bands, E2, E6 and E10 that were found in the resistant strains. The potency of the synergists piperonyl butoxide (PBO) and dihydrodillapiole (DDA) as esterase inhibitor were also studied both in vitro and in vivo. The in vitro results clearly show that both PBO and DDA inhibited esterase activity in the two resistant strains, while there was almost no esterase inhibition in the homogenate of the susceptible strain. The in vivo inhibition studies (topical application of PBO and DDA followed by biochemical analysis) illustrated that PBO- and DDA-esterase binding is rather slow and non permanent process. Esterase inhibition did not occur immediately after the synergist treatment but at 4 and 8 h post treatment in case of PBO and DDA, respectively. Native PAGE revealed that the in vivo esterase inhibition caused by both PBO and DDA was due to the binding of the synergist with the E6 isozyme which was not present in the susceptible strain.     

山东省绿盲蝽田间种群对六种杀虫剂的敏感性监测

为了解山东地区绿盲蝽对常用杀虫剂的敏感性变化情况,于2010—2012年采用玻璃管药膜法监测山东聊城、菏泽、滨州、德州地区绿盲蝽田间种群对马拉硫磷、毒死蜱、灭多威、丁硫克百威、联苯菊酯和氟虫腈等6种杀虫剂的敏感性。结果显示,相对于2009年的监测数据,2010—2012 年各地绿盲蝽种群对不同杀虫剂表现出不同程度的敏感性变化,但相对毒力比值均小于10倍。其中对毒死蜱、联苯菊酯的敏感性均未降低,菏泽种群表现为敏感性增强,相对毒力比值小于1。2011—2012年德州种群对丁硫克百威、灭多威和氟虫腈的敏感性降低,相对毒力比值大于3倍,其它种群敏感性变化不大,相对毒力比值均小于3倍。3年间菏泽种群对马拉硫磷的敏感性变化不大,相对毒力比值小于3倍,其它种群敏感性均有所降低。因此,毒死蜱、马拉硫磷、联苯菊酯、丁硫克百威等仍是山东棉区防治绿盲蝽的有效药剂。     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

棉铃虫对Cry1Ac抗性的稳定性及其对适合度的影响

为明确棉铃虫Helicoverpa armigera（Hübner）对苏云金芽胞杆菌Bacillus thuringiensis（Bt）Cry1Ac毒蛋白抗性的稳定性及其适合度变化,利用生物测定的方法研究了Cry1Ac抗性品系棉铃虫转到正常饲料饲养后的抗性衰退及再次筛选后抗性的恢复情况,并比较了敏感、抗性和抗性衰退后各品系间的适合度差异。在失去选择压的情况下,高抗品系棉铃虫对Cry1Ac的抗性迅速衰退,经过4代后抗性水平由最初的3626.67倍下降到1436.67倍;到第12代时抗性水平已低于10倍,随后品系保持较稳定的低抗水平;当重新进行抗性再筛选时,其抗性水平可快速恢复,抗性倍数快速回升,5代后恢复到1123.33倍。与敏感品系相比,高抗棉铃虫品系的适合度明显降低,相对适合度仅为0.33,但转到正常饲料连续饲养14代后,棉铃虫适合度明显上升,相对适合度为0.87,主要表现为卵孵化率和幼虫存活率等显著提高。     

Culex pipiens pallens: identification of genes differentially expressed in deltamethrin-resistant and -susceptible strains

Insecticide-resistance is a major obstacle to controlling insect vectors of microorganisms that cause human diseases. Identification of genes associated with resistance to insecticides has been a valuable tool for understanding mechanisms underlying resistance to commonly used insecticides such as deltamethrin. To identify such genes, we used suppression subtractive hybridization to obtain 809 differentially expressed clones in deltamethrin resistant versus susceptible laboratory strains of Culex pipiens pallens. Using cDNA microarrays and reverse Northern blots, a subset of 16 clones was confirmed to have greater than 3-fold difference in expression levels. Within this subset, we identified 2 clones uniquely expressed in the deltamethrin-resistant strain, eight clones exhibiting higher expression in the resistant strain and six in the susceptible strain. Of these 16 clones, 13 clones have sequence homology to known genes, such as ribosomal RNA, ribosome proteins, trypsin, and chymotrypsin-like proteins. Our data suggests resistance to deltamethrin may be a polygenic phenotype.     

Pyrethroid resistance and esterase activity in three strains of the cotton bollworm, Helicoverpa armigera (Hübner)

Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Azinphos-methyl and carbaryl resistance in adults of the codling moth (Cydia pomonella (L.), Lepidoptera: Tortricidae) from Northeastern Spain

The resistance of Cydia pomonella (L.) to organophosphates is widespread throughout the pome fruit growing areas. The lethal effects of two insecticides inhibitors of the acetylcholine esterase, azinphos-methyl and carbaryl, were evaluated in adults of five and four field populations of the codling moth, respectively. The lethal concentrations (LC₅₀ and LC₉₀) of these insecticides were determined in a susceptible strain from Spain (S_Spain). Topical bioassays using the approximate LC₉₀ values (3000 mg (a.i.)/L of carbaryl and 2000 mg (a.i.)/L of azinphos-methyl) that were obtained in S_Spain were tested as diagnostic concentrations. The enzymatic activities of mixed-function oxidases (MFO), glutathione S-transferases (GST) and esterases (EST) were measured to investigate their potential role in the detoxification of these insecticides.Carbaryl and azinphos-methyl caused ?53% and ?39% corrected mortality, respectively, in field populations, although the diagnostic concentrations applied were twofold and fourfold higher than the maximum concentration registered in Spain, respectively. The activities of MFO and GST were 7.3- to 16.1-fold higher and 2.5- to 3.7-fold higher in all the field populations compared to those in S_Spain, respectively.