Pyrethroid and carbamate resistance in Czech populations of Myzus persicae (Sulzer) from oilseed rape.

BACKGROUND: Failures in controlling Myzus persicae by pyrethroids and carbamates have been observed in Czechia since 2018. Eleven populations collected from Czech oilseed rape fields during 2018-2021 were tested for susceptibility to 11 insecticides. The presence of a single nucleotide polymorphism (SNP) leading to knockdown resistance in M. persicae populations was screened using allelic discriminating quantitative real-time polymerase chain reaction (qPCR). The presence of mutations related with the resistance of M. persicae to pyrethroids and carbamates was detected by sequencing paratype voltage-gated sodium channel and acetylcholinesterase 2 genes, respectively. RESULTS: Resistance to alpha-cypermethrin and pirimicarb was detected in most of the tested populations. The L1014F mutation was detected in 44.5% of M. persicae individuals surviving the field-recommended dose of alpha-cypermethrin. Sequencing of partial para gene for paratype voltage-gated sodium channel detected five different SNPs leading to four amino acid substitutions (kdr L1014F; s-kdr M918L; s-kdr M918T; and L932F). No pyrethroid-sensitive genotype was detected. The S431F amino acid substitution conferring resistance to carbamates was detected in 11 of 20 individuals with different pyrethroid-resistance genotypes. CONCLUSION: Resistance of M. persicae to both pyrethroids and carbamates was detected in nine of 11 populations. High resistance of M. persicae was correlated with mutations of the sodium channel. Sulfoxaflor, flonicamid, and spirotetramat are proposed as effective compounds to control pyrethroid- and carbamate-resistant populations of M. persicae. © 2023 Society of Chemical Industry.

Six-year monitoring of pesticide resistance in the Colorado potato beetle (Leptinotarsa decemlineata Say) during a neonicotinoid restriction period.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important potato pest with known resistance to pyrethroids and organophosphates in Czechia. Decreased efficacy of neonicotinoids has been observed in last decade. After the restriction of using chlorpyrifos, thiacloprid and thiamethoxam by EU regulation, growers seek for information about the resistance of CPB to used insecticides and recommended antiresistant strategies. The development of CPB resistance to selected insecticides was evaluated in bioassays in 69 local populations from Czechia in 2017-2022 and in 2007-2022 in small plot experiments in Zabcice in South Moravia. The mortality in each subpopulation in the bioassays was evaluated at the field-recommended rates of insecticides to estimate the 50% and 90% lethal concentrations (LC50 and LC90, respectively). High levels of CPB resistance to lambda-cyhalothrin and chlorpyrifos were demonstrated throughout Czechia, without significant changes between years and regions. The average mortality after application of the field-recommended rate of lambda-cyhalothrin was influenced by temperature before larvae were sampled for bioassays and decreased with increasing temperature in June. Downwards trends in the LC90 values of chlorpyrifos and the average mortality after application of the field-recommended rate of acetamiprid in the bioassay were recorded over a 6-year period. The baseline LC50 value (with 95% confidence limit) of 0.04 mg/L of chlorantraniliprole was established for Czech populations of CPBs for the purpose of resistance monitoring in the next years. Widespread resistance to pyrethroids, organophosphates and neonicotinoids was demonstrated, and changes in anti-resistant strategies to control CPBs were discussed.

Bias in sex ratios and polyandry rate in reproduction of Leptinotarsa decemlineata.

The Colorado potato beetle (CPB, Leptinotarsa decemlineata Slechtd.) is an invasive pest with economic importance worldwide. Sex ratios during egg-hatching and a frequency of polyandry in single-female families were analysed to clarify the reproduction strategy of CPB, which was still known only in fragments. 1296 just hatching 1st instar CPB larvae were collected from 19 single-female families, of which 13 were random families collected from potato fields and 6 were families produced by laboratory farming of naturally fertilised females. All larvae were analysed to detect a sex using a qPCR-based method and to detect polymorphisms in genotypes of 9 microsatellite (SSR) markers. The bias in sex ratio in favour of females was confirmed using linear mixed-effects model in both experimental groups of families: field collections (F = 36.39; P = 0.0001) and laboratory farming (F = 13.74; P = 0.0139). The analysis of diversity in microsatellites proved the polyandry in all progenies as 73% of analysed segregation patterns did not match with the patterns expected for full-sib progenies; on average per locus, 46% of allelic and 49.7% of genotype ratios showed irregular segregation. Both findings contribute toward understanding CPB success rate as an invasive species, as the preferential bearing of females with polyandry has a great potential to keep fitness of progenies, to maintain and operate population diversity, and to accelerate the reproduction of the pest.

Proteogenomic insight into the basis of the insecticide tolerance/resistance of the pollen beetle Brassicogethes (Meligethes) aeneus.

The pollen beetle is a major pest of oilseed rape. Although various resistance mechanisms have been identified, such as kdr (mutation in the sodium channel) and metabolic resistance (CYP overexpression), other "hidden" factors also exist. Some studies have stressed the importance of epistasis as a genetic background. The combination of kdr and metabolic resistance appears to be unfavorable under field conditions in the absence of pesticide selection. The regulation of detoxification enzymes can play an important role, but we highlight different detoxification markers compared to those emphasized in other studies. We also stress the importance of studying the role of markers identified as pathogenesis-related protein 5-like (PR5; upregulated by insecticides) and highlight the role of RNA (DEAD-box) helicases (downregulated by insecticides). Thus, we suggest the importance of epigenetic drivers of resistance/tolerance to pesticides. The key results are similar to those of our previous study, in which deltamethrin treatment of the pollen beetle was also investigated by a proteogenomic approach. Indeed, the mechanism leading to resistance of the pollen beetle may be an innate mechanism that the pollen beetle can also employ in natural habitats, but under field conditions (pesticide exposure), this mechanism is used to survive in response to insecticides. SIGNIFICANCE: Pesticide resistance is a serious problem that hampers the successful production of crops. Understanding the mechanisms of insecticide resistance is highly important for successful pest control, especially when considering integrated pest management. Here, using a proteogenomic approach, we identified novel markers for understanding pollen beetle resistance to pesticides. In addition, future studies will reveal the role of these markers in the multiresistance of pollen beetle populations. We highlight that the proteins identified as PR5, which are known to occur in beetles and are similar to those in plants, may be responsible for tolerance to multiple stresses. In addition, our results indicate that the RNA helicases that exhibited changes in expression may be the epigenetic drivers of multiresistance. The nature of these changes remains an open question, and their relevance in different situations (responses to different stresses) in natural habitats in the absence of pesticides can be proposed.

Evaluation of Pesticide Residue Dynamics in Lettuce, Onion, Leek, Carrot and Parsley.

The dynamics of 32 active substances contained in pesticide formulations (15 fungicides and 17 insecticides) were analyzed in iceberg lettuce, onion, leek, carrot, and parsley. Pesticide residues were monitored from the time of application until harvest. In total, 114 mathematical models of residue dissipation were developed using a first-order kinetic equation. Based on these models, it was possible to predict the action pre-harvest interval (the time between the last pesticide application and crop harvest) needed to attain a targeted action threshold (value significantly lower than the maximum limit) for low-residue vegetable production. In addition, it was possible to determine an action pre-harvest interval based on an action threshold of 0.01 mg kg-1 to produce vegetables intended for zero-residue production. The highest amount of pesticide residues were found in carrot and parsley leaves several days after treatment, and pesticide dissipation was generally slow. Lower amounts were found in leeks and lettuce, but pesticide dissipation was faster in lettuce. According to our findings, it seems feasible to apply reduced pesticide amounts to stay below unwanted residue levels. However, understanding the effectivity of reduced pesticide application for controlling relevant pest organisms requires further research.

Cabbage stem flea beetle's (Psylliodes chrysocephala L.) susceptibility to pyrethroids and tolerance to thiacloprid in the Czech Republic.

The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala (Coleoptera: Chrysomelidae), has recently become a major pest species in winter oilseed rape in the Czech Republic. The susceptibility of CSFB populations from two localities to six pyrethroids, two neonicotinoids, one organophosphate and one oxadiazine was evaluated in 2015-2018 in glass vial experiments. The susceptibility of CSFB to thiacloprid and thiamethoxam was evaluated in feeding experiment in 2017 and 2018. High susceptibility of CSFB populations to lambda-cyhalothrin, cypermethrin, esfenvalerate, tau-fluvalinate, etofenprox, deltamethrin, chlorpyrifos, indoxacarb and acetamiprid was observed in the glass vial experiments. The LC50 and LC90 data obtained for pyrethroids in these experiments in 2015 represent baseline for CSFB resistance monitoring to pyrethroids in the Czech Republic. High tolerance of CSFB to thiacloprid of CSFB was demonstrated in glass vial and the feeding experiment, too.

Seven-year monitoring of pyrethroid resistance in the pollen beetle (Brassicogethes aeneus F.) during implementation of insect resistance management.

BACKGROUND: An increase in the spread of pyrethroid resistance in the pollen beetle has been documented in many European countries. Pyrethroid resistance in the pollen beetle in the Czech Republic has been detected using a topical application bioassay. Resistance monitoring has been carried out during the implementation of insect resistance management. RESULTS: The susceptibilities of nine pollen beetle populations to four pyrethroids in 2009 - 2015 in the Czech Republic are presented in this paper. The highest resistance ratio (RR) values [based on the lethal dose for 50% of the population (LD50 )] obtained for deltamethrin, lambda-cyhalothrin, tau-fluvalinate and etofenprox were 500, 299, 108 and 66.9, respectively. Pollen beetle mortality after application of deltamethrin or lambda-cyhalothrin gradually decreased from 2009 to 2013. High cross-resistance between lambda-cyhalothrin and deltamethrin and low cross-resistance between lambda-cyhalothrin and etofenprox were demonstrated. A kdr mutation known to cause resistance in the pollen beetle was not detected. CONCLUSION: A high level of resistance was recorded in most of the nine populations of the pollen beetle in 2013. In the following 2 years, a decrease in resistance associated with a population density decrease of the pollen beetle was observed in all nine populations as a result of insect resistance management based on pyrethroid reduction in winter oilseed rape. © 2017 Society of Chemical Industry.

Beyond the survival and death of the deltamethrin-threatened pollen beetle Meligethes aeneus: An in-depth proteomic study employing a transcriptome database.

Insecticide resistance is an increasingly global problem that hampers pest control. We sought the mechanism responsible for survival following pyrethroid treatment and the factors connected to paralysis/death of the pollen beetle Meligethes aeneus through a proteome-level analysis using nanoLC coupled with Orbitrap Fusion™ Tribrid™ mass spectrometry. A tolerant field population of beetles was treated with deltamethrin, and the ensuing proteome changes were observed in the survivors (resistant), dead (paralyzed) and control-treated beetles. The protein database consisted of the translated transcriptome, and the resulting changes were manually annotated via BLASTP. We identified a number of high-abundance changes in which there were several dominant proteins, e.g., the electron carrier cytochrome b5, ribosomal proteins 60S RPL28, 40S RPS23 and RPS26, eIF4E-transporter, anoxia up-regulated protein, 2 isoforms of vitellogenin and pathogenesis-related protein 5. Deltamethrin detoxification was influenced by different cytochromes P450, which were likely boosted by increased cytochrome b5, but glutathione-S-transferase ε and UDP-glucuronosyltransferases also contributed. Moreover, we observed changes in proteins related to RNA interference, RNA binding and epigenetic modifications. The high changes in ribosomal proteins and associated factors suggest specific control of translation. Overall, we showed modulation of expression processes by epigenetic markers, alternative splicing and translation. Future functional studies will benefit. BIOLOGICAL SIGNIFICANCE: Insects develop pesticide resistance, which has become one of the key issues in plant protection. This growing resistance increases the demand for pesticide applications and the development of new substances. Knowledge in the field regarding the resistance mechanism and its responses to pesticide treatment provides us the opportunity to propose a solution for this issue. Although the pollen beetle Meligethes aeneus was effectively controlled with pyrethroids for many years, there have been reports of increasing resistance. We show protein changes including production of isoforms in response to deltamethrin at the protein level. These results illustrate the insect's survival state as a resistant beetle and in its paralyzed state (evaluated as dead) relative to resistant individuals.

Evaluation of pesticide residue dynamics in Chinese cabbage, head cabbage and cauliflower.

Pesticide residues from the time of application until harvest were analysed for 20, 17 and 18 active insecticidal and fungicidal substances in Chinese cabbage, head cabbage and cauliflower, respectively. In total, 40 mathematical models of residue degradation were developed using a first-order kinetic equation, and from these models it was possible to forecast the action pre-harvest interval for a given action threshold for low-residue production in Brassica vegetables as a percentage of the maximum residue level. Additionally, it was possible to establish an action pre-harvest interval based on an action threshold of 0.01 mg kg‒1 for the production of Brassica vegetables for baby food. Among the evaluated commodities, the speed of residue degradation was highest in head cabbage, medium in Chinese cabbage and lowest in cauliflower. The half-lives of pesticide in various vegetables were also determined: they ranged from 1.55 to 5.25 days in Chinese cabbage, from 0.47 to 6.54 days in head cabbage and from 1.88 to 7.22 days in cauliflower.

Inhibitory specificity and insecticidal selectivity of alpha-amylase inhibitor from Phaseolus vulgaris.

The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.

Detection of organophosphate and pyrethroid resistance alleles in Czech Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) populations by molecular methods.

BACKGROUND: Pyrethroids and organophosphates are the most frequently used insecticides for Colorado potato beetle, Leptinotarsa decemlineata (Say), control in the Czech Republic. Based on molecular methods, organophosphate and pyrethroid resistance alleles have been detected in samples from three sites. The accuracy of restriction fragment length polymorphism (RFLP) and bidirectional polymerase chain reaction amplification of specific alleles (Bi-PASA) for detection of resistance alleles is compared. RESULTS: Leptinotarsa decemlineata from three sites showed higher frequencies of resistance alleles to organophosphates than to pyrethroids. The rates of occurrence of individuals homozygous resistant (RR) to pyrethroids ranged from 20.0 to 22.9%, while the rates of occurrence of individuals RR to organophosphates ranged from 52.9 to 66.7%. The incidences of individuals with resistance alleles to both organophosphates and pyrethroids ranged from 8.6 to 13.6%. No relationship was found between incidence of the pyrethroid resistance allele and site, while incidence of the organophosphate resistance allele differed significantly according to site. CONCLUSION: Both RFLP and Bi-PASA were suitable for detecting resistance alleles to pyrethroids, and in most cases also for detecting resistance alleles to organophosphates. In contrast to Bi-PASA, RFLP was also suitable for samples with lower DNA quality when testing for the resistance allele to pyrethroids. On the other hand, RFLP was not as accurate as Bi-PASA in detection of the organophosphate resistance allele.