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.     

Cuticular penetration of β-cypermethrin in insecticide-susceptible and resistant strains of Bactrocera dorsalis

Semi-thin sectioning and transmission electron microscope techniques were employed to investigate the cuticle thickness, integument structure, and fat body of larvae from susceptible and resistant strains of Bactrocera dorsalis. The results showed that the cuticle of β-cypermethrin-resistant strains (25.96 ± 1.00 μm) was thicker than that of susceptible strains (19.36 ± 0.82 μm). The number of chitin layers in the endocuticle of β-cypermethrin-resistant strains (98.00 ± 3.61 layers) was more than that in susceptible strains (75.67 ± 2.40 layers). Compared with susceptible strains, the laminated structure of the chitin layers in the endocuticle of resistant strains revealed higher density and more distinctive structure, and the interspace of epidermal cells was thicker. Fat body in the resistant insects contained more fat granules than those in susceptible insects. Moreover, HPLC analysis showed that the cuticular penetration of β-cypermethrin into larvae of resistant strains was slower than that of susceptible strains. In addition, the metabolism of β-cypermethrin in resistant strains was faster than that in susceptible strains, indicating that the resistant strains could enhance detoxification metabolism. These results indicated that cuticle thickness, fat body, laminated structure of the chitin layers, and interspace of epidermal cells might be correlated with cuticular penetration between susceptible and resistant strains, suggesting that the resistant strains could decrease the rate of penetration of insecticide into the internal cavity.     

三唑磷与氟虫腈复配对粘虫的增效机理研究

以粘虫Mythimna separate 4龄幼虫为试虫,从表皮穿透、生物转化、作用靶标3个方面研究了三唑磷·氟虫腈复配剂的增效机理。结果表明,三唑磷与氟虫腈复配后,相互促进了对方对粘虫表皮的穿透作用;三唑磷对酸性磷酸酯酶(ACPase)的竞争性抑制与氟虫腈对ACPase的非竞争性抑制与反竞争性抑制的混合型抑制相结合,使复配剂对ACPase的抑制能力大幅度提高;对谷胱甘肽S-转移酶(GSTs)的联合抑制系数(JIC)达到425.87,表现出显著的增效作用;三唑磷和氟虫腈对乙酰胆碱酯酶(AChE)只有微弱的交互作用。表皮穿透率的提高及其对ACPase和GSTs抑制能力的增强可能是该复配剂对粘虫增效的重要机理。     

The mechanisms of carbaryl resistance in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

The physiological mechanisms of resistance to carbaryl were investigated in a carbaryl-resistant strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl butoxide greatly reduced the resistance level from 90- to 6-fold, indicating that microsomal cytochrome P-450-dependent monooxygenases may play a major role in resistance. This finding is consistent with metabolic data in which the oxidative metabolism of carbaryl by midgut homogenates was five times more active in the resistant strain than in the susceptible strain. In addition, the resistant strain showed increased activities of microsomal hydroxylation and epoxidation compared to the susceptible strain. Cuticular penetration studies using [¹⁴C]carbaryl revealed that 55% of the applied radioactivity remained on the cuticle of resistant larvae while 32% remained on susceptible larvae 24 hr after topical treatment. The resistance appeared to be unrelated to target site insensitivity. It is concluded that the high level of resistance to carbaryl in this insect was mainly due to enhanced oxidative metabolism of the insecticide (via hydroxylation and epoxidation) with reduced cuticular penetration playing a very minor role, if any.     

Mechanism of resistance to spinosyn in the tobacco budworm, Heliothis virescens

Topical laboratory selection of tobacco budworm larvae, Heliothis virescens, with technical spinosad for multiple generations resulted in larvae 1068-fold resistant to topical applications of the insecticide and 316.6-fold resistant to insecticide treated diet as compared to the parental strain. The penetration of 2′-O-methyl[¹⁴C]spinosyn A across the cuticle of the susceptible (parental) and selected (resistant) tobacco budworms increased with time 3-12 h after application. A trend of reduced penetration in the resistant strain was found but the differences were not statistically significant. 2′-O-methyl[¹⁴C]spinosyn A when injected into the hemocoel was not metabolized 96 h after treatment in both the susceptible and resistant strain, suggesting that a change in metabolism was not the mechanism of resistance. Electrophysiological studies indicated that dose-dependent spinosyn A-induced currents occurred in neurons from spinosyn resistant and susceptible (adult) tobacco budworms. At both 10 and 100 nM spinosyn A, however, the amplitude of these currents in the resistant insects was significantly smaller than the amplitude of currents observed from neurons from susceptible tobacco budworm adults. This suggests that neurons from resistant insects have decreased sensitivity to spinosyn A. However, the reduced inward currents in the resistant strain may or may not be related to the mode of action of the spinosyns. No statistically significant cross-resistance was noted for the spinosad resistant tobacco budworms for topical applications of permethrin (Pounce^®), profenofos (Curacron^®), emamectin benzoate (Denim^®), or indoxacarb (Steward^®). A statistically significant reduction in susceptibility to acetamiprid (Mospilan^®) in artificial diet as determined from a resistance ratio of 0.482 was found.     

Resistance in the highly DDT-resistant 91-R strain of Drosophila melanogaster involves decreased penetration,increased metabolism,and direct excretion

Resistance to 4,4′-dichlorodiphenyltrichloroethane (DDT) in the 91-R strain of Drosophila melanogaster is extremely high compared to the susceptible Canton-S strain (>1500 times). In addition to enhanced oxidative detoxification, the 91-R strain also has a reduced rate of DDT penetration, increased levels of reductive and conjugative metabolism, and substantially more excretion than the Canton-S strain. Contact penetration of DDT was ∼30% less with 91-R flies, which also had significantly more cuticular hydrocarbons and a thicker, more laminated cuticle compared to Canton-S flies, possibly resulting in penetration differences. DDT was metabolized ∼1.6-fold more extensively by 91-R than Canton-S flies, resulting in dichlorodiphenyldichloroethane (DDD), two unidentified metabolites and polar conjugates being formed in significantly greater amounts. 91-R flies also excreted ∼4-fold more DDT and metabolites than Canton-S flies. Verapamil pretreatment reduced the LD₅₀ value for 91-R flies topically dosed with DDT by a factor of 10-fold, indicating that the increased excretion may involve, in part, ATP-binding cassette (ABC) transporters. In summary, DDT resistance in 91-R is polyfactorial and includes reduced penetration, increased detoxification and direct excretion.     

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 characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in the resistant and susceptible strains (SS) of Liposcelis bostrychophila were investigated. The two resistant strains were the dichlorvos-resistant strain (DDVP-R) and the phosphine-resistant strain (PH₃-R) with resistance ratios of 22.36 and 4.51, respectively. Compared to their susceptible counterpart, the AChE activity per insect and the specific activity of AChE in DDVP-R and PH₃-R were significantly higher. There were also significant kinetic differences between DDVP-R and PH₃-R. The apparent Michaelis-Menten constant (K_m) for acetylthiocholine iodide (ATChI) was obviously lower in SS than that in PH₃-R, indicating a higher affinity to the substrate ATChI in the susceptible strains. The affinity for the substrate ATChI in DDVP-R and SS were not significantly different. The V_max value of the PH₃-R was significantly greater when compared to the V_max for the SS suggesting a possible over expression of AChE in this resistant strain. The inhibition of AChE to insecticide exposure in vitro revealed that all six insecticides were inhibitory for the extracted AChE’s. Based on the I₅₀ values, AChE of the SS were more sensitive to dichlorvos, paraoxon-ethyl, malaoxon and demeton-S-methyl than those of the two resistant strains. As for carbaryl and eserine, the PH₃-R suggested a significantly higher I₅₀s compared to the susceptible strain, while, no significant differences were found between SS and DDVP-R.     

Cry1Ac和Cry2Ab蛋白对大草蛉生长发育及酶活力的影响

为明确转Cry1Ac和Cry2Ab基因棉花对大草蛉的影响,运用Bt蛋白与人工饲料混合的方法,以大于转基因棉花叶片中蛋白含量20倍的剂量饲喂大草蛉初孵幼虫,初步研究了Cry1Ac和Cry2Ab对大草蛉生物学参数和消化酶、解毒酶活性的影响。结果表明：取食含Bt蛋白饲料的大草蛉幼虫的发育历期、体重、蛹重、成虫体重、羽化率等生物学参数与对照相比均没有显著差异;在大草蛉幼虫体内可以检测到含量较高的Cry1Ac和Cry2Ab蛋白,分别为974.92~1 282.39 ng/g鲜重和5 592.62~6 082.92 ng/g鲜重,而在大草蛉成虫体内检测到的Cry1Ac和Cry2Ab蛋白含量非常低,分别为0.29~0.39 ng/g鲜重和50.34~56.71 ng/g鲜重;取食含Bt蛋白的饲料对大草蛉幼虫的类胰蛋白酶、类胰凝乳蛋白酶、氨肽酶和谷胱甘肽-S-转移酶活性有一定的影响,但对大草蛉成虫影响与对照差异不显著。表明大草蛉取食含Bt蛋白的人工饲料后,虽然体内可以检测到一定含量的Bt蛋白,但对大草蛉的生长发育并没有显著的直接不利影响。     

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.     

Fitness cost, cross resistance and realized heritability of resistance to imidacloprid in Spodoptera litura (Lepidoptera: Noctuidae)

Imidacloprid has been used as a key insecticide for controlling sucking insect pests of cotton, whereas Spodoptera litura also has been indirectly exposed to this insecticide in Pakistan. To evaluate the risk of resistance evolution and to develop a better resistance management strategy, a field collected population was selected with imidacloprid in the laboratory. Thereafter, fitness cost, realized heritability and cross resistance of imidacloprid resistance in S. litura were investigated. After 14 generations of selection with imidacloprid, S. litura developed a 137.48-fold resistance to the insecticide. Bioassay revealed that this strain showed cross-resistance to acetamiprid (RR 8.52) and a little to lamdacyhalothrin (1.92) but negative cross-resistance was found to methomyl (−0.19). The resistant strain had a relative fitness of 0.38, with substantially lower rates of larval survival, larval duration, male pupal duration, development time, emergence rate of healthy adults, fecundity, hatchability, and prolonged larval and pupal duration. Mean relative growth rate of the larvae, intrinsic rate of population increase, and biotic potential was lower for the selected populations. The estimated realized heritability (h²) of imidacloprid resistance was 0.15 in the resistant strain of S. litura. Development of the resistance may cost significant fitness for the resistant population. This study provided valuable information for further understanding the impact of imidacloprid resistance on physiological parameters of S. litura and for facilitating the development of resistance management strategies.     

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.     

Synthesis and insecticidal activities of pyridine ring derivatives of podophyllotoxin

In an attempt to find the biorational pesticides, we synthesized 12 pyridinyl derivatives of podophyllotoxin (PPT) and 4′-demthylepipodophylltoxin (4′-DMEP) in this study. Their structures and the α/β substitution at C-4 were confirmed by ¹H NMR, IR, MS spectral analyses and elemental analysis. The insecticidal activities were tested against fifth-instar larvae of Pieris rapae and the third-instar larvae of Cullex pipiens pallens at concentrations of 250 and 10 μg ml⁻¹. Four derivatives of PPT, 4.1, 4.2, 4.3 and 4.5, showed higher insecticidal activities against P. rapae than PPT, while three derivatives of PPT, 4.4, 4.5 and 4.6, displayed higher mosquito larvicidal activity than PPT, with LC₅₀ values of 1.66, 3.96 and 1.54 mg l⁻¹, respectively. Interestingly, we also found that the pyridine ring derivatives of PPT showed delayed insecticidal activity, which is different from traditional neurotic insecticides. The results suggest that 4′-OCH₃ in the PPT derivatives is essential to keep the insecticidal activity and the insecticidal activities of pyridine ring derivatives of PPT are higher than that of the derivatives of 4′-DMEP, supporting PPT has the potential to be a lead structure of semi-synthetic insecticides.     

Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera)

Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three ¹²⁵I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no significant difference in Cry1Ac-binding affinity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (R_t decreased from 5.87 ± 1.40 to 2.23 ± 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance fitted the “mode 1” pattern of Bt resistance described previously.     

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH₃-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I₅₀ value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.     

Mode of action of the plant-derived silphinenes on insect and mammalian GABAA receptor/chloride channel complex

The silphinenes are tricyclic sesquiterpenes that have antifeedant and toxic effects in insects and structural similarity to the known GABA antagonist, picrotoxinin. In murine synaptoneurosomes, silphinenes block GABA-stimulated influx of ³⁶Cl⁻ with EC₅₀s in the range of 10-30 μM. In insects, silphinenes were tested in neurophysiological recordings of central neurons from third instar Drosophila melanogaster larvae. Silphinenes reversed the blockage of neuronal firing induced by GABA, but had little effect below 100 μM. The structure-activity profile observed in the murine chloride flux assay was also observed in the larval neurophysiological assay, indicating little selectivity for the silphinenes. A reference silphinene was equally active on nerve preparations from the rdl strain of D. melanogaster, which is resistant to channel-blocking antagonists via an altered GABA receptor. This latter finding suggests that silphinenes interact with the insect GABA receptor in a manner somewhat different from PTX, and that rdl resistance in the field may have little effect on silphinene efficacy.     

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。