Effect of dihydrodillapiole on pyrethroid resistance associated esterase inhibition in an Indian population of Spodoptera litura (Fabricius)

Resistance in Spodoptera litura (Fabricius) has been attributed to enhanced detoxification of insecticides by increased levels of esterases, oxidases and/or glutathione S-transferases. Enzyme inhibiting insecticide synergists can be employed to counter increased levels of such enzymes in S. litura. Dihydrodillapiole induced synergism of pyrethroid toxicity was examined in the laboratory-reared third instar larval population of S. litura collected in Delhi (susceptible), and Guntur (resistant) region of Andhra Pradesh, India. The Guntur population was found to be 7.04 and 10.19 times resistant to cypermethrin and lambdacyhalothrin, respectively. The activity of cypermethrin, lambdacyhalothrin and profenophos against susceptible and resistance populations of S. litura, was gradually increased when used along with a plant-derived insecticide synergist dihydrodillapiole. The α-naphthyl acetate hydrolysable esterase activity in Delhi population was less as compared to the Guntur population. Resistance associated esterases in Delhi population were inhibited by pre-treatment with dihydrodillapiole. The esterase level in insect was instantly reduced initially, sustained for about 3 h and equilibrated at 4 h post treatment. The esterase activity of Guntur population was increased to 1.28 μmoles/mg/min at 2 h post treatment and subsequently reduced to lower than 0.70 μmoles at 4-12 h post treatment. The variation in esterase activity is suggestive of its homeostatic regulation in test populations. Dihydrodillapiole thus caused significant reduction of resistance in S. litura to cypermethrin, lambda cyhalothrin and profenophos.     

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.     

Partial purification and characterization of a methyl-parathion resistance-associated general esterase in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Increased hydrolytic metabolism of organophosphate insecticides has been associated with resistance among Nebraska western corn rootworm populations. In this study, resistance-associated esterases were partially purified by differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor of 100-fold and recovery of approximately 10%. Kinetic analysis of the partially purified enzyme indicated that the K_m of the group II esterases was identical for the two populations, although V_max was consistently threefold higher in the resistant population. A putative esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII is a monomer with a molecular weight of approximately 66 kDa, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. Immunoassays with the Myzus persicae E4 antiserum indicated that group II esterases from D. v. virgifera were cross-reactive and expressed at much higher titers in the resistant population relative to the susceptible counterpart. These results suggest that the resistance is likely associated with overproduction of an esterase isozyme in resistant D. v. virgifera populations.     

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.     

Cloning of the acetylcholinesterase 1 gene and identification of point mutations putatively associated with carbofuran resistance in Nilaparvata lugens

Molecular mechanisms of carbofuran resistance in the brown planthopper, Nilaparvata lugens Stål, were investigated. A carbofuran-resistant strain (CAS) showed approximately 45.5- and 15.1-fold resistance compared with a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Activities of the esterase and mixed-function oxidase were approximately 2.8- and 1.6-fold higher, respectively, in the CAS strain than in the SUS strain, suggesting that these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was approximately 5.5- and 3.7-fold higher in the CAS strain compared to the SUS and FM strains, respectively, indicating that AChE insensitivity is associated with carbofuran resistance. Western blot analysis identified two kinds of AChEs, of which the type-1 AChE (encoded from Nlace1, which is paralogous to the Drosophila AChE gene) was determined to be the major catalytic AChE in N. lugens. The open reading frame of Nlace1 is composed of 1989 bp (approximately 74 kD) and revealed 52.5% and 24.3% amino acid sequence identities to those of Nephotettix cincticeps and Drosophila melanogaster, respectively. Screening of point mutations identified four amino acid substitutions (G119A, F/Y330S, F331H and H332L) in the CAS strain that likely contribute to AChE insensitivity. The frequencies of these mutations were well correlated with resistance levels, confirming that they are associated with reduced sensitivity to carbofuran in N. lugens. These point mutations can be useful as genetic markers for monitoring resistance levels in field populations of N. lugens.     

哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达

采用室内筛选与田间防效相结合的方法,对哈茨木霉抑制黄瓜尖孢镰刀菌的拮抗机制进行了研究。对峙培养结果显示,木霉菌和病原菌间均形成了较明显的抑菌圈,对病原菌的抑菌率达66.7%~85.8%,其中菌株TG、TM对枯萎病菌抑制作用较强。木霉菌可有效提高根系对病原菌的抗性,黄瓜植株接种枯萎病菌后,根系细胞大量死亡,而先接种木霉菌再接种病原菌后则减小了对根系的伤害。田间防效试验结果表明,TG和TM孢子悬浮液浓度在108个/mL时防效最好,分别为54.9%和49.4%。接种木霉菌植株根系抗性基因的表达量均高于对照植株,呈双峰趋势。在第6天时抗性基因表达量最高,WRKY6、MYB、PR-1、PAL、GST和GLU的表达量分别为对照的5.15、5.22、6.07、6.00、3.16、和16.15倍。表明木霉菌通过激活与胁迫相关的基因表达提高了对病原菌的抗性。     

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.     

Expression of Ace-paralogous acetylcholinesterase of Culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system

In Ace paralogous acetylcholinesterase (AP-AChE) of Culex tritaeniorhynchus, an amino acid substitution, Phe455Trp, is accompanied by the insecticide insensitivity. To confirm the responsibility of the substitution to the insensitivity, AP-AChE cDNA with and without a Phe455Trp substitution and Ace orthologous AChE (AO-AChE) cDNA were expressed in a baculovirus-insect cell system and the biochemical properties of AChEs (AP-CxTI, AP-CxTS, and AO-CxT, respectively) determined. AP-CxTI which has the same level of affinity to ACh, the natural substrate, showed a drastic decline in affinity to the artificial substrates composed of a longer moiety. The sensitivity of AP-CxTI to inhibitors was extremely reduced when compared with AP-CxTS. The insensitivity to tested organophosphates was greater than to monomethyl carbamates. AO-AChE showed similar substrate specificity and a slightly higher sensitivity to inhibitors when compared with AP-CxTS. Taking the position of Phe455Trp in the acyl pocket of the active site into account, the dimensions of the acyl pocket appear to became smaller by the substitution and insensitive to inhibitors.     

Combined detoxification mechanisms and target mutation fail to confer a high level of resistance to organophosphates in Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Despite the frequent and widespread applications of organophosphates against Cydia pomonella this species has developed low levels of resistance to this chemical group. Investigations concerning the mechanisms involved in resistance are scarce, and usually consider only one of the potential mechanisms. With the aim of a better understanding the resistance mechanisms and their possible interaction, four of these mechanisms were investigated simultaneously in one sensitive (Sv) and two resistant strains (Raz and Rdfb) of this insect. Resistant strains displayed an increased mixed function oxidase activity, whereas carboxylesterase activity varied upon the substrate used. The three strains had similar β-naphtyl acetate activity, and the hydrolysis of α-naphthyl acetate and p-nitrophenyl valerate was higher in the Sv strain. The p-nitrophenyl acetate activity was highest in the resistant strains and was strongly inhibited by azinphos and DEF. The Raz strain has a modified acetylcholinesterase (AChE), which resulted in a 0.7-, 3.2- and 21.2-fold decrease in the susceptibility to chlorpyriphos-ethyl-oxon, azinphos-methyl-oxon, and paraoxon-methyl, respectively. These combined resistance mechanisms only conferred to Raz a 0.6-, 7.9- and 3.1-fold resistance to the related insecticides. Organophosphates resistance in C. pomonella results from a combination of mechanisms including modified affinities to carboxylesterase substrates, and increased metabolisation of the insecticide. The apparent antagonism between increased functionalisation and reduced sensitivity of the AChE target is discussed.     

First molecular genotyping of voltage gated sodium channel alleles in Culex quinquefasciatus populations in Malaysia

A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.     

草莓镶脉病毒ORF Ⅵ基因的原核表达与抗血清制备

为了分析草莓镶脉病毒（Strawberry vein banding virus,SVBV）ORF Ⅵ基因的功能,采用原核表达技术获得该基因表达的重组蛋白并制备其抗血清。利用PCR技术扩增得到SVBV中国分离物的ORF Ⅵ基因,将此基因克隆到原核表达载体pET-SUMO上,获得重组质粒pET-SUMO-ORF Ⅵ。转化大肠杆菌BL21（DE3）后,经IPTG诱导与Ni²⁺-NTA亲和柱纯化,获得分子质量约为90 kD的重组蛋白。以纯化的重组蛋白为抗原免疫家兔制备抗血清,采用间接ELISA和Western blot方法测定抗血清的效价、反应灵敏度以及ORF Ⅵ基因在本氏烟中的瞬时表达。结果显示,间接ELISA法测定抗血清对重组蛋白的效价达1：256 000,Western blot法能够检测到稀释64 000倍的重组蛋白。利用稀释2 000倍的抗血清,仍能够检测出SVBV中国分离物和美国分离物ORF Ⅵ基因在本氏烟中瞬时表达的蛋白。     

三叶草斑潜蝇hsp70的克隆及其表达量在高低温胁迫下的变化

为了明确高低温胁迫对三叶草斑潜蝇hsp70表达量的影响,采用RT-PCR和RACE技术获得了1条三叶草斑潜蝇诱导型热激蛋白基因hsp70,命名为Lthsp70-1,并利用实时荧光定量PCR技术检测其在温度胁迫后的表达量.该基因的开放阅读框为1923 bp,编码640个氨基酸.氨基酸序列中含有HSP70家族的签名序列IFDLGGGTFDVSIL和IVLVGGSTRIPK、DnaK特征基序IDLGTT(Y)S(C)V、非细胞器基序RARFEEL,以及C末端的保守序列EEVD.实时荧光定量PCR结果显示:成虫在31～33℃范围内,其hsp70表达量随温度升高而上升,33℃时达到最高峰;35～39℃时,hsp70表达量迅速下降;蛹经0℃胁迫0.5～2.0 h,其hsp70表达量随时间延长呈上升趋势.由此可见,高低温胁迫均能诱导三叶草斑潜蝇hsp70的表达.     

Single nucleotide polymorphism detection at the Hypothenemus hampeiRdl gene by allele-specific PCR amplification with Tm-shift primers

The resistant Rdl allele for dieldrin insecticide was detected on the Hypothenemus hampei populations from Colombia using conventional PCR methods. Based on this sequence, a melting temperature (T_m) shift genotyping method that relies on allele-specific PCR is described for insecticide resistance-associated single nucleotide polymorphism (SNP) at the H. hampeiRdl gene. The method reported here uses GC-rich tails of unequal length attached to allele-specific primers containing 3′ terminal bases that correspond to SNP allelic variants. Specific PCR products are identified by inspection of a melting curve on a real-time PCR thermocycler using SYBR Green DNA binding dye. Resistant and susceptible alleles resulted in specific PCR products with T_m of 83.3 ± 0.1 °C and 86.0 ± 0.2 °C, respectively. The RdlT_m-shift genotyping method is a new method to identify the Rdl gene in the coffee berry borer H. hampei, the principal pest of coffee that in general show low genetic diversity and very few genetic strategies for control of this pest have been developed. The method supplies a high-throughput tool for dieldrin resistance-associated SNP diagnostic in the coffee berry borer which will be useful for resistance-management strategies and as genetic marker in the colombian insect populations for genetics research.     

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.     

Development of high-throughput real-time PCR assays for the identification of insensitive acetylcholinesterase (ace-1) in Anopheles gambiae

Resistance to the organophosphate and carbamate insecticides through insensitivity of the target site enzyme, acetylcholinesterase has recently been reported in Anopheles gambiae populations in West Africa. To date, screening for the mutation (G119S of the ace-1 gene) conferring this insensitivity has employed a simple PCR-RFLP diagnostic. However, this has the disadvantage of requiring digestion of the amplified fragment and subsequent gel electrophoresis of the products. To overcome this, and thus increase throughput and reduce costs, we have developed two assays based on real-time PCR (TaqMan and melt-curve) that represent true ‘closed-tube’ approaches. The two new platforms were compared to PCR-RFLP to genotype over 280 samples. The two new methods compared favourably with PCR-RFLP with the TaqMan assay delivering the greatest specificity and sensitivity of the three approaches. This assay is also cheaper to run than PCR-RFLP and results are obtained in a single step.     

Molecular basis for resistance to tribenuron in shepherd’s purse (Capsella bursa-pastoris (L.) Medik.)

Capsella bursa-pastoris, a winter annual weed in the mustard family, can not be controlled by tribenuron after the herbicide has been continuously used for several years. The resistant biotype Lz-R was the generation of a population collected from Liangzhu, a place where tribenuron had been used for more than 15 consecutive years. To confirm and characterize the resistance of C. bursa-pastoris to tribenuron, whole-plant bioassays were conducted in the greenhouse. The results of whole-plant bioassays revealed that Lz-R was highly resistant to tribenuron with the resistance index (GR₅₀ Lz-R)/(GR₅₀ Lz-S) up to 236.6. To investigate the molecular basis of resistance in C. bursa-pastoris, the acetolactate synthase (ALS) genes were sequenced and compared between susceptible and resistant biotypes. Analysis of the nucleotide and deduced amino acid sequences between the biotypes indicated that one substitution had occurred in Domain A, cytosine by thymine (CCT to TCT) at position 197, that led to a change of the amino acid proline in the susceptible to serine in the Lz-R.