A comparative study of insecticide resistance assays with the German cockroach

Females of a diazinon-resistant strain of German cockroach exhibiting cross resistance to propoxur were evaluated for resistance to diazinon and propoxur by six assays: LD₅₀s for topical application and injection; LC₅₀, KT₅₀ and LT₅₀ for deposits on wood and KT₅₀ for deposits on glass. Also investigated were certain variables in the topical application technique and the experimental conditions for topical synergism studies. Among all assays, diazinon resistance ratios (resistant:susceptible) ranged from 3- to 145-fold; propoxur varied from 1- to 14-fold. Resistance ratios based on KT₅₀ data were consistently low (<4x) for both insecticides. Resistance to diazinon was 13x when applied topically and 6x when injected. Resistance to propoxur was 14x by injection compared to 8x for topical application. The LC₅₀ assay exhibited the greatest difference in resistance to the two insecticides: namely, 145x to diazinon and 1x(no resistance) to propoxur. By LT₅₀ analysis, resistance to propoxur was 10x compared with 100x resistance to diazinon. Thus, the method used had a significant effect on the final value of the resistance ratio. Even though resistance ratios may vary widely among different techniques, the final choice of a method depends upon many factors. The LT₅₀ method may provide a more realistic appraisal of resistance in wild populations of German cockroaches especially when experimental residues are similar to those used in control programmes.     

Pyrethroid resistance and cross‐resistance in the German cockroach,Blattella germanica (L)

A German cockroach (Blatella germanica (L)) strain, Apyr‐R, was collected from Opelika, Alabama after control failures with pyrethroid insecticides. Levels of resistance to permethrin and deltamethrin in Apyr‐R (97‐ and 480‐fold, respectively, compared with a susceptible strain, ACY) were partially or mostly suppressed by piperonyl butoxide (PBO) and S,S,S,‐tributylphosphorotrithioate (DEF), suggesting that P450 monooxygenases and hydrolases are involved in resistance to these two pyrethroids in Apyr‐R. However, incomplete suppression of pyrethroid resistance with PBO and DEF implies that one or more additional mechanisms are involved in resistance. Injection, compared with topical application, resulted in 43‐ and 48‐fold increases in toxicity of permethrin in ACY and Apyr‐R, respectively. Similarly, injection increased the toxicity of deltamethrin 27‐fold in ACY and 28‐fold in Apyr‐R. These data indicate that cuticular penetration is one of the obstacles for the effectiveness of pyrethroids against German cockroaches. However, injection did not change the levels of resistance to either permethrin or deltamethrin, suggesting that a decrease in the rate of cuticular penetration may not play an important role in pyrethroid resistance in Apyr‐R. Apyr‐R showed cross‐resistance to imidacloprid, with a resistance ratio of 10. PBO treatment resulted in no significant change in the toxicity of imidacloprid, implying that P450 monooxygenase‐mediated detoxication is not the mechanism responsible for cross‐resistance. Apyr‐R showed no cross‐resistance to spinosad, although spinosad had relatively low toxicity to German cockroaches compared with other insecticides tested in this study. This result further confirmed that the mode of action of spinosad to insects is unique. Fipronil, a relatively new insecticide, was highly toxic to German cockroaches, and the multi‐resistance mechanisms in Apyr‐R did not confer significant cross‐resistance to this compound. Thus, we propose that fipronil could be a valuable tool in integrated resistance management of German cockroaches. © 2001 Society of Chemical Industry     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae)

Resistance to insecticides remains a major problem for the successful control of the horn fly, Haematobia irritans irritans (L.), one of the most important pests of cattle in many countries including the United States. The organophosphate (OP) insecticide diazinon has been used to control pyrethroid-resistant populations of the horn fly. There are only a few reported cases of horn fly resistance to diazinon in the United States and Mexico. Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides to control horn flies. PBO-synergized diazinon products are also available for horn fly control in the United States, although PBO is known to inhibit the bio-activation of certain OP insecticides including diazinon. A study was conducted to evaluate the effect of PBO on diazinon toxicity to horn flies using a filter paper bioassay technique. These bioassays in both the susceptible and diazinon-resistant horn fly strains revealed a biphasic effect of PBO on diazinon toxicity to horn flies. PBO inhibited diazinon toxicity when the PBO concentration used was high (5%), and no effect was observed when PBO concentration was intermediate (2%). However, at low concentrations (1% and lower), PBO significantly synergized diazinon toxicity. We demonstrated that enhanced esterase activity was associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases from this study suggest that PBO did not have significant effect on the overall esterase activity in the horn fly. The observed synergistic effect of PBO at lower concentrations on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. It is likely that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the cuticle and/or inhibiting the oxidative detoxification of diazinon, and reduced diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.     

Correlation of a resistance-associated Rdl mutation in the German cockroach, Blattella germanica (L), with persistent dieldrin resistance in two Danish field populations

Danish Blattella germanica (L) populations carry the resistance-associated mutation A302S within the Resistance to dieldrin (Rdl) gene. The mutation has remained in field populations long after the discontinuation of dieldrin for cockroach control. The mutation has also persisted in our laboratory strains with high and intermediate frequencies for more than 8 years without selection. The toxicity of dieldrin was tested by topical application to male cockroaches in the susceptible strain DPIL-SUS and two field strains, Zo960302 and Su960304, which were 1270- and 15-fold resistant to dieldrin at LD50. We report the sequencing of exon 7 of the B. germanica Rdl gene and the finding of the putative resistance-associated A302S mutation. We have developed and implemented a PCR-based diagnostic method with the detection of a restriction endonuclease polymorphism, which allows for easy discrimination of susceptible, resistant and heterozygote genotypes. The frequency of the resistance-associate allele A302S was 0.97 and 0.38 in the Zo960302 and Su960304 populations, respectively. The cockroach Rdl A302S allele confers high dieldrin resistance in homozygotes and intermediate resistance in heterozygotes, and its presence is responsible for the persisting dieldrin resistance in Danish populations of B. germanica.     

Development of strategies for monitoring indoxacarb and gel bait susceptibility in the German cockroach (Blattodea: Blattellidae)

BACKGROUND: Advion^® cockroach gel bait (6 g kg^?1 indoxacarb) is in widespread use for Blattella germanica (L.) control in the United States. However, baseline susceptibility levels to indoxacarb in German cockroach field populations are not known. Hence, this research sought to develop monitoring strategies to estimate the susceptibility of German cockroach populations to indoxacarb. RESULTS: Four bioassays were evaluated: topical lethal dose (LD), formulated gel bait lethal time (LT), vial lethal concentration (LC) and gel bait matrix LD. Of these methods, the vial LC and gel bait matrix LD bioassays were the most economical and relevant assessment strategies. For indoxacarb susceptibility monitoring, a two‐tiered approach was developed that utilizes diagnostic concentrations and doses in vial LC (30 and 60 µg vial^?1) and gel bait matrix LD (1.0, 1.5 and 2.5 µg insect^?1) formats. CONCLUSIONS: A two‐tiered susceptibility monitoring strategy was developed that includes testing field populations at diagnostic concentrations and doses in first‐tier vial LC bioassays and second‐tier gel bait matrix LD bioassays. The vial method facilitates rapid identification of field strains with reduced susceptibility. The feeding bioassay effectively simulates field exposure to Advion^® and therefore has utility for secondary confirmation of susceptibility shifts and identification of behavioral resistance (i.e. bait aversion). Copyright © 2010 Society of Chemical Industry     

Various mechanisms responsible for permethrin metabolic resistance in seven field-collected strains of the German cockroach from Iran, Blattella germanica (L.) (Dictyoptera: Blattellidae)

Insecticide resistance in the German cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism. Seven field-collected strains of German cockroach, Blattella germanica (L.) with various levels of resistance to pyrethroids, five out of which were also cross-resistant to DDT were used in this study. The investigation of possible mechanisms responsible for permethrin resistance was carried out using the synergists PBO, DEF and DMC and biochemical assays, including general esterases, glutathione S-transeferases and monooxygenases assays, using an automated microtitre plate reader. PBO and DEF, the inhibitors of cytochrome p450 monooxygenases and general esterases, respectively, affected permethrin resistance to varying degrees depending on the strain. DDT resistance in five strains were not completely eliminated by the synergist DMC, an inhibitor of glutathione S-transferase enzymes, suggesting that a further non-metabolic resistance mechanism such as kdr-type may be present. This suggestion was further supported by GST assay data, where a little elevation in GST activity was detected in only two strains. The synergist data supported by biochemical assays implicated that cytochrome p450 monooxygenases or hydrolases are involved in permethrin resistance in some strains. However, these results implicated both enhanced oxidative and hydrolytic metabolism of permethrin as resistance mechanism in the other strains. The results of synergist and biochemical studies implicated that all the field-collected permethrin resistant strains have developed diverse mechanisms of resistance, although these strains have been collected from the same geographic area. The change in resistance ratios of some strains by using PBO or DEF is discussed. It is of interest to note that because resistance to permethrin was not completely eliminated by DEF and PBO, it is likely that one or more additional mechanisms are involved in permethrin resistance in every strain studied.     

Efficacy of noviflumuron gel bait for control of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae)-laboratory studies

The insecticidal activity of a cockroach gel bait containing a chitin synthesis inhibitor, noviflumuron, was evaluated using laboratory and field strains of the German cockroach, Blattella germanica (L.). Noviflumuron gel bait (0.01-5 mg g(-1)) caused > or = 90% nymphal mortality to laboratory and field strains of B. germanica in choice tests after 11 and 19 days of continuous exposure respectively. In 1 m x 1 m bioassay arenas, laboratory strain B. germanica population levels exposed to 5 mg g(-1) noviflumuron bait or 0.1 mg g(-1) fipronil gel bait were significantly lower than untreated population levels after 3 weeks and 1 week of exposure respectively. Various noviflumuron bait exposure periods (2, 4 and 7 weeks) caused similar population reductions, with a mean of 99.3 (+/- 0.3)% at 7 weeks. Fipronil gel bait caused 100% population reduction at 2 weeks post-exposure. The control population increased 89.0% at 7 weeks. In a simulated kitchen experiment with mixed stage laboratory populations, cockroach trap catches decreased 96.8 (+/- 2.0)% at 8 weeks in the 0.5 mg g(-1) noviflumuron bait treatment. The trap catches in the control increased 506.5 (+/- 493.7)% during the same period. Trap catch reduction by 0.1 mg g(-1) fipronil gel bait reached 100% at 4 weeks. Noviflumuron bait caused significantly lower nymph/total ratios to B. germanica populations in bioassay arenas from 2 weeks after exposure, demonstrating its effectiveness as a control agent for B. germanica with a pattern of activity similar to that expected from a chitin synthesis inhibitor.     

Phenotype and spectrum of action of the Pvr4 resistance in pepper against potyviruses, and selection for virulent variants

The dominant Pvr4 gene identified in Capsicum annuum cv. Criollo de Morelos 334 (CM334) is frequently used in pepper cultivars because it possesses one of the largest spectra of action among plant virus resistance genes. This gene was previously shown to confer efficient resistance to all known Potato virus Y isolates, to Pepper mottle virus , to Pepper yellow mosaic virus and to Ecuadorian rocoto virus. This study showed that the W4 line, derived from CM334 and carrying Pvr4 , was also resistant to Peru tomato mosaic virus and Pepper severe mosaic virus , but not to Pepper veinal mottle virus , Chilli veinal mottle virus or Tobacco etch virus . It was noticed that the phenotype of the resistance was atypical since, in the W4 line, hypersensitive reaction or extreme resistance could be observed, depending on virus isolates and inoculated organs. Despite the large deployment of Pvr4 in hybrid cultivars, the numerous tests performed in controlled conditions and the use of W4 serial back-inoculations with potyvirus isolates controlled by this line, no virulent variant isolates were obtained. However, it was shown that the use of graft inoculation experiments allow PVY virulent variants to be selected.     

Synergism of diazinon toxicity and inhibition of diazinon metabolism in the house fly by tridiphane: Inhibition of glutathione S-transferase activity

Diazinon toxicity to a susceptible strain of house fly (Musca domestica L.) was synergized by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane], a herbicide synergist. Both diazinon and tridiphane were partially metabolized in the house fly by glutathione (GSH) conjugation. Synergism appeared to be due to inhibition of diazinon metabolism/detoxification. Crude glutathione S-transferase (GST) preparations from the house fly catalyzed GSH conjugation of diazinon, tridiphane, 3,4-dichloronitrobenzene (DCNB), and chloro-2,4-dinitrobenzene (CDNB). Tridiphane and the GSH conjugate of tridiphane appeared to inhibit diazinon GSH conjugation, but diazinon did not inhibit tridiphane GSH conjugation. The enzymatic rate of tridiphane GSH conjugation was 22 times the rate of diazinon GSH conjugation; therefore, attempts to assay tridiphane as an inhibitor of diazinon GSH conjugation were inconclusive because of the high concentration of tridiphane GSH conjugate produced during the assay. CDNB underwent enzymatic GSH conjugation at a rate 240 times faster than that of tridiphane and 5000 times faster than that of diazinon. GSH conjugation of CDNB was not inhibited by tridiphane, but was inhibited by the GSH conjugate of tridiphane. In vivo, the GSH conjugate of tridiphane was produced in sufficient concentration to cause the observed inhibition of diazinon metabolism and synergism of diazinon toxicity. However, the possibility that parent tridiphane caused or contributed to the inhibition of diazinon metabolism and synergism of diazinon toxicity could not be excluded. Inhibition of diazinon metabolism did not appear to be due to depletion of either GSH or GST.     

桔全爪螨对甲氰菊酯的抗性选育及交互抗性的研究

采集桔全爪螨敏感种群 ,在室内用甲氰菊酯处理 ,以选育其抗药性。结果显示 :选育至 12代 ,抗性达 17 11倍 ,且发展速度越来越快。交互抗性测定结果显示 ,甲氰菊酯抗性品系对功夫和哒螨灵有非常明显的交互抗性 ,对卡死克有非常明显的负交互抗性现象。     

Boric acid toxicity to the German cockroach, Blattella germanica: Alterations in midgut structure, and acetylcholinesterase and glutathione S-transferase activity

Oral toxicity of boric acid, an inorganic insecticide, was evaluated on German cockroach, Blattella germanica L. (Dictyoptera, Blattellidae). Newly emerged adults were exposed to various concentrations of boric acid incorporated into the diet. Results showed that treated insects exhibited toxic symptoms with a dose-dependent mortality. Histological study of midgut revealed alterations in the epithelial cells and a significant increase in the epithelium thickness. In a second series of experiments, the compound was investigated on the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs). Data showed that the compound induced GSTs and reduced the activity of AChE. From this experiment, it may be concluded that ingested boric acid caused death of insects perhaps ultimately by starvation via alterations of the midgut. Additionally, the compound seemed to present a neurotoxic action as evidenced by the symptoms of poisoning and the reduction in AchE activity.     

Genetic analysis and effect of synergists on diazinon resistance in the bulb mite,rhizoglyphus rohini claparède (acari:Acaridae)

A genetic analysis revealed that the resistance to diazinon of the bulb mite, Rhizoglyphus robini Claparède. lacked sex linkage, and was based on the operation of a dominant allele. Synergism of diazinon by eight inhibitors of detoxifying enzymes in susceptible and resistant strains of the bulb mite was determined. Esterases were shown to play an important role in detoxification against diazinon. S.S.S-tributyl phosphorotrithioate, an esterase inhibitor, was the only synergist which obviously synergized diazinon. The others, including the inhibitors of glutathione-S-transferases, carboxylesterase and mixed function oxidases, showed obscure or even antagonistic effect.     

Buprofezin susceptibility survey, resistance selection and preliminary determination of the resistance mechanism in Nilaparvata lugens (Homoptera: Delphacidae)

BACKGROUND: Buprofezin has been used for many years to control Nilaparvata lugens (Stål). Assessment of susceptibility change in the insect is essential for maintaining control efficiency and resistance management. RESULTS: Eleven‐year surveys showed that most field populations were susceptible before 2004. However, substantially higher levels of resistance (up to 28‐fold) were found in most of the rice fields in China after 2004. A field population was collected and periodically selected for buprofezin resistance in the laboratory. After 65 generations (56 were selected), the colony successfully obtained 3599‐fold resistance to buprofezin. Synergism tests showed that O,O‐diethyl‐O‐phenyl phosphorothioate (SV1), piperonyl butoxide (PBO) and diethyl maleate (DEM) increased buprofezin toxicity in the resistant strain by only 1.5–1.6 fold, suggesting that esterases, P450‐monooxygenases and glutathione S‐transferases had no substantial effect on buprofezin resistance development. CONCLUSION: The results from this study indicate that N. lugens has the potential to develop high resistance to buprofezin. A resistance management program with rotation of buprofezin and other pesticides may efficiently delay or slow down resistance development in the insect. Further investigation is also necessary to understand the resistance mechanisms in N. lugens. Copyright © 2008 Society of Chemical Industry