Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).

BACKGROUND: Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase-based mechanism has been attributed to pyrethroid resistance and over-expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. RESULTS: In vitro interactions between putative synergists and CYP6CM1, B and Q-type esterases were investigated, and structure-activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B-type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q-esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. CONCLUSIONS: Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industry.

The interactions of piperonyl butoxide and analogues with the metabolic enzymes FE4 and CYP6CY3 of the green peach aphid Myzus persicae (Hemiptera: Aphididae).

BACKGROUND: Piperonyl butoxide (PBO) is a well-known insecticide synergist capable of interacting with phase 1 metabolic enzymes, specifically esterases and cytochrome P450s. In this study, structure-activity relationship analyses were used to characterise the interaction of around 30 analogues of PBO with the esterase FE4 and the P450 CYP6CY3 from insecticide-resistant Myzus persicae (Sulzer), in order to predict the synthesis of more potent inhibitors. RESULTS: Enzyme inhibition studies were performed against esterase and oxidase activities and, together with in silico modelling, key activity determinants of the analogues were identified and optimised. Novel analogues were then designed and synthesised, some of which showed greater inhibition against both enzymatic systems: specifically, dihydrobenzofuran moieties containing an alkynyl side chain and a butyl side chain against FE4, and benzodioxole derivatives with a propyl/butyl side chain and an alkynyl ether moiety for CYP6CY3. CONCLUSIONS: In vitro assays identified potential candidate synergists with high inhibitory potency. The in vivo confirmation of such results will allow consideration for a possible use in agriculture. © 2016 Society of Chemical Industry.

The use of substituted alkynyl phenoxy derivatives of piperonyl butoxide to control insecticide-resistant pests.

BACKGROUND: Derivatives of piperonyl butoxide with alkynyl side chains were tested in vitro and in vivo against pyrethroid-resistant Meligethes aeneus and imidacloprid-resistant Myzus persicae. RESULTS: Synergists with the alkynyl side chain were more effective inhibitors of P450 activity in vitro than piperonyl butoxide, and demonstrated high levels of synergism in vivo, with up to 290-fold synergism of imidacloprid against imidacloprid-resistant M. persicae. CONCLUSIONS: These 'second-generation' synergists could overcome metabolic resistance in many pest species and possibly enable reduced rates of insecticide application in some cases. © 2016 Society of Chemical Industry.

Synergistic manipulations of plant and insect defences.

BACKGROUND: It has been demonstrated previously that cis-jasmone acts as an elicitor of plant defence mechanism(s) by inducing secondary metabolism. It has also been demonstrated that temporal synergism can result in hypersensitive insect pests due to the inhibition of metabolic enzymes. RESULTS: Laboratory bioassays demonstrated that pre-exposure of insects by piperonyl butoxide followed by cis-jasmone treatment of crops, reduced Aphis gossypii on cotton by 80% and Myzus persicae on sweet pepper by 90%. By microencapsulating the cis-jasmone and combining with piperonyl butoxide, Bemisia tabaci on tomatoes was reduced by 99%. A field trial with microencapsulated cis-jasmone combined with piperonyl butoxide resulted in a comparable reduction of whitefly egg numbers to that given by the registered rate of imidacloprid, with efficacy of 89% and 93%, respectively. CONCLUSIONS: If insect defence enzymes are compromised by piperonyl butoxide whilst plant defence is primed by cis-jasmone, there are possibilities of an insecticide-free method of controlling insect pests. The success seems largely dependent upon the toxicity of the plants' secondary chemistry.

The interactions between piperonyl butoxide and E4, a resistance-associated esterase from the peach-potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae).

BACKGROUND: It has been reported previously that piperonyl butoxide (PBO) can inhibit both P450 and esterase activity. Although the method by which PBO combines with cytochrome P450 has been identified, the way in which it acts as an esterase inhibitor has not been established. This paper characterises the interactions between PBO and the resistance-associated esterase in Myzus persicae, E4. RESULTS: After incubation with PBO/analogues, hydrolysis of 1-naphthyl acetate by E4 is increased, but sequestration of azamethiphos is reduced. Rudimentary in silico modelling suggests PBO docks at the lip of the aromatic gorge. CONCLUSIONS: PBO binds with E4 to accelerate small substrates to the active-site triad, while acting as a blockade to larger, insecticidal molecules. Structure-activity studies with analogues of PBO also reveal the essential chemical moieties present in the molecule.

Repellents inhibit P450 enzymes in Stegomyia (Aedes) aegypti.

The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.

The effect of a piperonyl butoxide/tau-fluvalinate mixture on pollen beetle (Meligethes aeneus) and honey bees (Apis mellifera).

BACKGROUND: Previous work has characterised pyrethroid resistance in pollen beetle (Meligethes aeneus F.) as principally an oxidative mechanism. Piperonyl butoxide (PBO) can synergise this resistance in the field, but its effects on the honey bee are thought to be unacceptable. RESULTS: A field trial in Poland was conducted to show that a mixture of PBO and tau-fluvalinate at the registered rate gave increased and longer-lasting control of resistant pollen beetle. Four days after spraying with tau-fluvalinate, only 20% of pollen beetles were controlled, compared with 70% if the tau-fluvalinate/PBO mixture was used. No detriment to honey bee health was observed using the same mixture. CONCLUSIONS: PBO, if used in conjunction with a pyrethroid of relatively low bee toxicity, can successfully overcome pyrethroid resistance in pollen beetle without incurring an increased loss of honey bees, even if they are present at the time of spraying.

Investigating the potential of selected natural compounds to increase the potency of pyrethrum against houseflies Musca domestica (Diptera: Muscidae).

BACKGROUND: A study was undertaken to determine the efficacy of seven natural compounds compared with piperonyl butoxide (PBO) in synergising pyrethrum, with the intention of formulating an effective natural synergist with pyrethrum for use in the organic crop market. RESULTS: Discriminating dose bioassays showed PBO to be significantly more effective at synergising pyrethrum in houseflies than the seven natural compounds tested, causing 100% mortality in insecticide-susceptible WHO and resistant 381zb strains of housefly. The most effective natural synergists against WHO houseflies were dillapiole oil, grapefruit oil and parsley seed oil, with 59, 50 and 41% mortality respectively, compared with 18% mortality with unsynergised pyrethrum. Against 381zb houseflies, the most effective natural synergists were parsley seed oil and dillapiole oil. Esterase inhibition by the natural compounds and PBO in vitro showed no correlation with pyrethrum synergism in vivo, whereas the inhibition of oxidases in vitro more closely correlated with pyrethrum synergism in vivo. CONCLUSION: Dillapiole oil and parsley seed oil showed the greatest potential as pyrethrum synergists. PBO remained the most effective synergist, possibly owing to its surfactant properties, enhancing penetration of pyrethrins. The results suggest the involvement of oxidases in pyrethroid resistance in houseflies, with the efficacy of synergists showing a high correlation with inhibition of oxidases.

Characterising metabolic resistance in pyrethroid-insensitive pollen beetle (Meligethes aeneus F.) from Poland and Switzerland.

BACKGROUND: Pollen beetle (Meligethes aeneus F.) has become the most important pest of oilseed rape in Europe, but its control has been greatly hindered by pyrethroid resistance. Target-site resistance has been implicated previously, and, whilst synergism has been found with piperonyl butoxide (PBO), the exact nature of metabolic resistance has remained unknown. The use of PBO, in conjunction with its analogue EN 16/5-1, has allowed the characterisation of metabolic resistance. RESULTS: In vitro assays in combination with in vivo studies using PBO and EN 16/5-1 showed that high synergism of pyrethroids was primarily correlated with an oxidative mechanism, although a limited contribution by esterases was implicated in one population. CONCLUSION: Differential synergism has enabled the characterisation of pyrethroid resistance in populations of M. aeneus. It was found to be principally due to an oxidative-based mechanism, and, if a synergist were to be used to inhibit this enzyme group, renewed control against resistant pests could be achieved.

Metabolic enzyme(s) confer imidacloprid resistance in a clone of Myzus persicae (Sulzer) (Hemiptera: Aphididae) from Greece.

BACKGROUND: Previous studies have reported varying levels of resistance against imidacloprid in several insect species, including populations of the peach-potato aphid, Myzus persicae (Sulzer). These cases of resistance have been attributed to either target-site resistance or enhanced detoxification. In this study, a clone of M. persicae originating from Greece revealed a 60-fold resistance factor to imidacloprid. RESULTS: The Greek clone is compared in terms of metabolic enzyme activity and synergism profiles with other M. persicae clones showing lower imidacloprid resistance. CONCLUSION: A combination of in vitro biochemical assays and in vivo differential synergism studies using PBO and a close analogue EN 16/5-1 suggests that the mechanism conferring increased resistance in this clone is primarily due to enhanced oxidase activity.

An analogue of piperonyl butoxide facilitates the characterisation of metabolic resistance.

BACKGROUND: Previous work has demonstrated that piperonyl butoxide (PBO) not only inhibits microsomal oxidases but also resistance-associated esterases. The ability to inhibit both major metabolic resistance enzymes makes it an ideal synergist to enhance xenobiotics but negates the ability to differentiate which enzyme group is responsible for conferring resistance. RESULTS: This study examines an analogue that retains the ability to inhibit esterases but is restricted in its ability to act on microsomal oxidases, thus allowing an informed decision on resistance enzymes to be made when used in conjunction with the parent molecule. CONCLUSION: Using examples of resistant insects with well-characterised resistance mechanisms, a combination of PBO and analogue allows identification of the metabolic mechanism responsible for conferring resistance. The relative potency of PBO as both an esterase inhibitor and an oxidase inhibitor is also discussed.

Esterase isoenzymes and insecticide resistance in Frankliniella occidentalis populations from the south-east region of Spain.

BACKGROUND: Frankliniella occidentalis (Pergande) is among the most important crop pests in the south-east region of Spain; its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. To this end, F. occidentalis populations, collected from the field at different locations in south-east Spain, were studied in terms of total esterase activity and esterase isoenzyme pattern. RESULTS: Individual thrips extracts were analysed by native polyacrylamide gel electrophoresis (PAGE) and stained for esterase activity with the model substrate alpha-naphthyl acetate. Significant correlations were found between resistance to the insecticides acrinathrin and methiocarb and the presence of a group of three intensely stained bands, named Triplet A. For each individual thrips extract, total esterase activity towards the substrates alpha-naphthyl acetate and alpha-naphthyl butyrate was also measured in a microplate reader. Insects possessing Triplet A showed a significantly higher alpha-naphthyl acetate specific activity and alpha-naphthyl acetate/alpha-naphthyl butyrate activity ratio. This observation allowed a reliable classification of susceptible or resistant insects either by PAGE analysis or by total esterase activity determination. CONCLUSION: The PAGE and microplate assays described can be used as a monitoring technique for detecting acrinathrin- and methiocarb-resistant individuals among F. occidentalis field populations.

A novel assay reveals the blockade of esterases by piperonyl butoxide.

BACKGROUND: Conventional in vitro assays sometimes fail to reveal esterase inhibition by piperonyl butoxide (PBO), although synergism studies suggest loss of esterase-mediated sequestration of insecticide does take place. A new in vitro assay has been devised that routinely reveals binding between PBO and these esterases. RESULTS: The new 'esterase interference' assay detects the blockade of resistance-associated esterases in a species, Myzus persicae Sülzer, where this has not previously been seen. The assay also demonstrates directly the protective effect esterases may confer to target sites of insecticides. CONCLUSION: The new assay reveals esterase blockade by PBO and thus has the potential to be used as a high-throughput screening method for other potential synergists.

Cross-resistance between a Bacillus thuringiensis Cry toxin and non-Bt insecticides in the diamondback moth.

BACKGROUND: Bacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides. RESULTS: Selection of a subpopulation of the P. xylostella SERD4 population with the pyrethroid deltamethrin increased resistance to both deltamethrin and Cry1Ac relative to an unselected subpopulation. Selection of a second subpopulation with the Bt toxin Cry1Ac also increased resistance to both Cry1Ac and deltamethrin. A complementation test between the Cry1Ac-selected and deltamethrin-selected subpopulations suggested the presence of a common genetic locus or loci that control resistance to both insecticides. A piperonyl butoxide analogue with potent inhibitory activity against insect esterases significantly increased the toxicity of Cry1Ac and deltamethrin against the respective resistant subpopulations, but showed no such synergism with the unselected subpopulation of SERD4. CONCLUSION: Selection of one resistance phenotype resulted in the simultaneous selection of the other. This phenomenon could be due to a single mechanism acting against both classes of insecticide or to genetically linked, but separate, mechanisms.

Temporal synergism can enhance carbamate and neonicotinoid insecticidal activity against resistant crop pests.

BACKGROUND: Piperonyl butoxide (PBO) effectively synergises synthetic pyrethroids, rendering even very resistant insect pests susceptible, provided a temporal element is included between exposure to synergist and insecticide. This concept is now applied to carbamates and neonicotinoids. RESULTS: A microencapsulated formulation of PBO and pirimicarb reduced the resistance factor in a clone of Myzus persicae (Sulzer) from >19 000- to 100-fold and in Aphis gossypii (Glover) from >48 000- to 30-fold. Similar results were obtained for a strain of Bemisia tabaci Gennadius resistant to imidacloprid and acetamiprid, although a second resistant strain did not exhibit such a dramatic reduction, presumably owing to the presence of target-site insensitivity and the absence of metabolic resistance. Synergism was also observed in laboratory susceptible insects, suggesting that, even when detoxification is not enhanced, there is degradation of insecticides by the background enzymes. Use of an analogue of PBO, which inhibits esterases but has reduced potency against microsomal oxidases, suggests that acetamiprid resistance in whiteflies is largely oxidase based. CONCLUSION: Temporal synergism can effectively enhance the activity of carbamates and neonicotinoids against resistant insect pests. Although the extent of this enhancement is dependent upon the resistance mechanisms present, inhibition of background enzymes can confer increased sensitivity against target-site resistance as well as increased metabolism. .

Use of pyrethroid analogues to identify key structural features for enhanced esterase resistance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

It has been reported previously that the major resistance mechanism to pyrethroid insecticides by the cotton bollworm Helicoverpa armigera (Hübner) in Australia is a consequence of overproduction of esterase isoenzymes. This paper reports structure-activity relationships that support such a view, based on in vivo bioassays conducted with a range of pyrethroid structures containing a variety of acid and alcohol moieties and the correlation with in vitro esterase inhibition assays against the same structures, and identifies the critical regions of the molecule with regard to esterase inhibition, and hence resistance. The implications of this work in terms of possible resistance management are evaluated and discussed.

Temporal synergism by microencapsulation of piperonyl butoxide and alpha-cypermethrin overcomes insecticide resistance in crop pests.

A microencapsulated formulation that gives a burst release of piperonyl butoxide (PBO) several hours before a burst release of a conventional pyrethroid can effectively overcome metabolic resistance in Bemisia tabaci Gennadius, Helicoverpa armigera (Hübner), Aphis gossypii Glover and Myzus persicae Sulzer. This increase in efficacy against resistant pests was reflected in a field trial against B. tabaci on cotton, eliminating the need for two treatments. The ratio between the active insecticide and the synergist was found to be crucial in reducing resistance factors.

Effect of pretreatment with piperonyl butoxide on pyrethroid efficacy against insecticide-resistant Helicoverpa armigera (Lepidoptera: Noctuidae) and Bemisia tabaci (Sternorrhyncha: Aleyrodidae).

Pyrethroid resistance in B-type Bemisia tabaci Gennadius and Australian Helicoverpa armigera Hübner field populations is primarily conferred by esterase isoenzymes which metabolise and sequester pyrethroid insecticides. It has been shown previously that pyrethroid resistance-associated esterases in H. armigera are inhibited by the insecticide synergist piperonyl butoxide (PBO) over a 22-h period. It is demonstrated here that similar inhibition can be obtained against B-type B. tabaci. Small-scale field trials showed excellent levels of pyrethroid control when insects were pretreated with PBO and then dosed with pyrethroid during the time of maximum esterase inhibition. These results demonstrate that PBO can restore pyrethroid efficacy in the field against both B-type B. tabaci and resistant H. armigera.

New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin.

In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.

The effect of piperonyl butoxide on pyrethroid-resistance-associated esterases in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

Pyrethroid resistance in field populations of Australian Helicoverpa armigera (Hübner) is primarily a consequence of the overproduction of esterase isoenzymes which metabolise and sequester pyrethroid insecticides. Biochemical studies have shown that pyrethroid-resistance-associated esterases in H armigera are inhibited by the insecticide synergist piperonyl butoxide (PBO). Esterase inhibition by PBO did not occur immediately after dosing, but exhibited maximum inhibition 3-4 h after dosage. Esterase activity subsequently recovered until full activity was restored by 24 h. Topical bioassays using a pre-treatment of PBO showed that maximum H armigera mortality was achieved with pre-treatment times corresponding to maximum esterase inhibition. These results demonstrated that, with correct temporal application, PBO can restore pyrethroid efficacy against H armigera. It would also be expected that restoration of efficacy with other conventional insecticides, currently compromised by esterase-based resistance mechanisms, would occur.