High concentrations of piperonyl butoxide (PBO) enhance toxicity of S-methoprene against the lesser grain borer, Rhyzopertha dominica.

Insect growth regulators (IGRs) have been playing a major role in the effective management of a range of stored product insect pests including species that have developed resistance to major groups of insecticides, such as organophosphates (OPs) and synthetic pyrethroids (SPs). In the present study, we evaluated the efficacy of S-methoprene alone and in combination with piperonyl butoxide (PBO), an adjuvant component of insecticides for synergy, against two strains, Lab-S (susceptible) and Met-R (Methoprene resistant) of an economically important pest species, the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). Adults of both Lab-S and Met-R strains were exposed to wheat treated with multiple concentrations of S-methoprene ranging from 0.001 to 0.01 and 10 to 60 mg/kg, respectively, alone and in combination with PBO. A variety of concentrations, including 0.27, 0.53, 0.80, and 1.07 g/kg, were evaluated for PBO. Mortality of adults and percent reduction in progeny were assessed after 14 and 65 days of treatment, respectively. As anticipated, the adult mortality rates of both strains were not significantly affected by S-methoprene alone. However, the number of progeny produced confirmed that the Met-R strain exhibited a high level of resistance to S-methoprene alone, with over 50 F1 progeny adults surviving in wheat treated with the maximal rate, 10 mg/kg. In contrast, the toxicity of S-methoprene was increased against the same resistant strain (Met-R), by 0.80 or 1.07 g/kg of PBO in combination treatment, resulting in a significant reduction in progeny numbers (25 adults per container). Although the tested concentrations of S-methoprene and PBO were well above the currently registered rate globally, our results highlight the fact that PBO enhances the toxicity of S-methoprene to some extent, reaffirming that the mode of action of the latter involves the inhibition of mixed-function oxidases (MFOs) and highlights the need for further research into developing potential binary or triplet formulations containing these two active ingredients (AIs).

Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

Insect cuticle: a critical determinant of insecticide resistance

Intense use of insecticides has resulted in the selection of extreme levels of resistance in insect populations. Therefore understanding the molecular basis of insecticide resistance mechanisms becomes critical. Penetration resistance refers to modifications in the cuticle that will eventually slow down the penetration of insecticide molecules within insects' body. So far, two mechanisms of penetration resistance have been described, the cuticle thickening and the altering of cuticle composition. Cuticular modifications are attributed to the over-expression of diversified genes or proteins, which belong to structural components (cuticular proteins mainly), enzymes that catalyze enzymatic reactions (CYP4G16 and laccase 2) or ABC transporters that promote cuticular translocation. In the present review we summarize recent studies and discuss future perspectives.

International workshop on insecticide resistance in vectors of arboviruses, December 2016, Rio de Janeiro, Brazil.

Vector-borne diseases transmitted by insect vectors such as mosquitoes occur in over 100 countries and affect almost half of the world's population. Dengue is currently the most prevalent arboviral disease but chikungunya, Zika and yellow fever show increasing prevalence and severity. Vector control, mainly by the use of insecticides, play a key role in disease prevention but the use of the same chemicals for more than 40 years, together with the dissemination of mosquitoes by trade and environmental changes, resulted in the global spread of insecticide resistance. In this context, innovative tools and strategies for vector control, including the management of resistance, are urgently needed. This report summarizes the main outputs of the first international workshop on Insecticide resistance in vectors of arboviruses held in Rio de Janeiro, Brazil, 5-8 December 2016. The primary aims of this workshop were to identify strategies for the development and implementation of standardized insecticide resistance management, also to allow comparisons across nations and across time, and to define research priorities for control of vectors of arboviruses. The workshop brought together 163 participants from 28 nationalities and was accessible, live, through the web (> 70,000 web-accesses over 3 days).

Cytochrome P450 associated with insecticideresistance catalyzes cuticular hydrocarbonproduction in Anopheles gambiae

The role of cuticle changes in insecticide resistance in the major malariavector Anopheles gambiae was assessed. The rate of internalization of14C deltamethrin was significantly slower in a resistant strain than in asusceptible strain. Topical application of an acetone insecticide formu-lation to circumvent lipid-based uptake barriers decreased the resis-tance ratio by ∼50%. Cuticle analysis by electron microscopy andcharacterization of lipid extracts indicated that resistant mosquitoeshad a thicker epicuticular layer and a significant increase in cuticularhydrocarbon (CHC) content (∼29%). However, the CHC profile andrelative distribution were similar in resistant and susceptible insects.The cellular localization and in vitro activity of two P450 enzymes,CYP4G16 and CYP4G17, whose genes are frequently overexpressedin resistant Anopheles mosquitoes, were analyzed. These enzymesare potential orthologs of the CYP4G1/2 enzymes that catalyze thefinal step of CHC biosynthesis in Drosophila and Musca domestica, re-spectively. Immunostaining indicated that both CYP4G16 and CYP4G17are highly abundant in oenocytes, the insect cell type thought to se-crete hydrocarbons. However, an intriguing difference was indicated;CYP4G17 occurs throughout the cell, as expected for a microsomalP450, but CYP4G16 localizes to the periphery of the cell and lies onthe cytoplasmic side of the cell membrane, a unique position for a P450enzyme. CYP4G16 and CYP4G17 were functionally expressed in insectcells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrateand thus has bona fide decarbonylase activity similar to that ofdmCYP4G1/2. The data support the hypothesis that the coevolutionof multiple mechanisms, including cuticular barriers, has occurred inhighly pyrethroid-resistant An. gambiae.