Pollen Beetle Populations Research Articles

Examining spray-induced gene silencing for pollen beetle control

Pollen beetles are a transcontinental threat to oilseed rape cultivation. Conventional insecticide use has resulted in resistance development, and can furthermore lead to losses of non-target organisms, including those that contribute to natural biological control of pollen beetle populations. Biosafe alternatives to synthetic pesticides must be explored. Double-stranded RNA (dsRNA) targeting pollen beetle, by the RNA interference (RNAi) mode of action, has been previously demonstrated under laboratory conditions. The present study examined, under a whole plant context, the potential for spray-induced gene silencing (SIGS) in pollen beetle. We confirmed translocation of sprayed dsRNA to later-emerging, non-sprayed dietary tissues of oilseed rape, as well as infiltration of sprayed dsRNA to the inner tissues of reproductive buds (pollen beetle’s food source). No effect on survival was observed. The inefficacy of SIGS observed here highlights the requirement for more effective SIGS approaches to pollen beetle management. The translocation of sprayed dsRNA within oilseed rape plants, and the non-significant yet detectable effect on target mRNA downregulation, together suggest potential for future optimization of a SIGS approach against pollen beetle.

Resistance developments in Estonia’s population of pollen beetles (Brassicogethes aeneus)

A total of 111 local pollen beetle populations were collected from both winter and spring oilseed rape fields, in the main oilseed growing regions of Estonia between 2015−2019. The objective was to analyse the insecticide-susceptibility of the pollen beetle population (in the form of Brassicogethes aeneus). The pollen beetle samples were tested for sensitivity to lambda-cyhalothrin, thiacloprid, and chlorpyrifos. The efficacy of the tested insecticides varied considerably by region. We observed a clear decrease in susceptibility to lambda-cyhalothrin and thiacloprid, but sensitivity to chlorpyrifos remained stable throughout the period between 2015 and 2019. Amongst the tested samples in that period, a total of 3% were classified as susceptible to lambda-cyhalothrin, 18% as moderately resistant, 70% as resistant, and 7% as highly resistant. In the case of thiacloprid, 21% of the samples were highly susceptible to the insecticide, 39% were susceptible, and 41% had reduced levels of susceptibility to the insecticide. The information which was presented tended to confirm the ongoing evolution of insecticide resistance in the B. aeneus population in Estonia, while also highlighting the importance of data-based decisions when optimising insecticide resistance management in the field.

Widespread resistance of pollen beetles to pyrethroids in Czechia with no evidence for kdr mutation

The pollen beetle Brassicogethes aeneus (Fabricius, 1775) (Coleoptera: Nitidulidae) is one of the most important pests in oilseed rape (Brassica napus L. var. oleifera Moench) in Czechia. Wide-spread resistance development to pyrethroids in pollen beetle populations was documented in many European countries, including Czechia. The susceptibilities of 10 local pollen beetle populations originating from 10 localities in Czechia to lambda-cyhalothrin, tau-fluvalinate, thiacloprid, indoxacarb and chlorpyrifos were evaluated in 2017–2020 using glass vial bioassays. A high level of resistance to lambda-cyhalothrin was recorded in all the evaluated populations of the pollen beetle. The proportion of the populations resistant to tau-fluvalinate varied between years from 30% to 60%, and in 2018 and 2020, all the tested populations were classified as resistant. Despite the high level of resistance to pyrethroids, a kdr mutation known to cause resistance in the pollen beetle was not detected in any of the 244 tested individuals. In 2017–2020, the first local populations of the pollen beetle with decreased susceptibility to thiacloprid occurred. The LD90 of thiacloprid exceeded the field application dose in 100% and 90% of populations in 2018 and 2020, respectively. All the tested populations were susceptible to indoxacarb and chlorpyrifos. Cross-resistance between lambda-cyhalothrin and thiacloprid and between tau-fluvalinate and thiacloprid was found using data from 2017 to 2020. No cross-resistance between lambda-cyhalothrin and tau-fluvalinate was detected.

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. SignificancePesticide 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.

The correlation between the susceptibilities to lambda-cyhalothrin and tau-fluvalinate in Czech and Slovak pollen beetle populations

The correlation between the susceptibilities to lambda-cyhalothrin and tau-fluvalinate in Czech and Slovak pollen beetle populations

Changes in resistance development in pollen beetle (Brassicogethes aeneus F.) to lambda-cyhalothrin, etofenprox, chlorpyrifos-ethyl, and thiacloprid in the Czech Republic during 2013–2017

Susceptibility of common pollen beetle (Brassicogethes aeneus Fabricius) to four insecticides was monitored at 14 locations in the Czech Republic during 2013–2017 on oilseed rape. Seventy local pollen beetle populations were tested for resistance to lambda-cyhalothrin, etofenprox, chlorpyrifos-ethyl, and thiacloprid. Resistance to lambda-cyhalothrin and etofenprox was determined with the recommended rates of 7.5 g a.i. ha−1 and 40 g a.i. ha−1, respectively. Resistance increased significantly over time only for lambda-cyhalothrin. Chlorpyrifos-ethyl at the recommended rate of 187.5 g a.i. ha−1 exceeded by 4 times the mean 90% lethal dose (LD90) value of 44.4 g a.i. ha−1, and mortality was 100% in all years. Thiacloprid had widely dispersed LD50 and LD90 values, as did lambda-cyhalothrin and etofenprox. Thiacloprid's recommended rate of 72 g a.i. ha−1 was substantially higher than mean LD90 values. Several subpopulations with reduced susceptibility to thiacloprid were, however, found in this study. No cross-resistance among lambda-cyhalothrin, etofenprox, chlorpyrifos, and thiacloprid was determined.

Limited genetic structure and demographic expansion of the Brassicogethes aeneus populations in France and in Europe.

The pollen beetle, Brassicogethes aeneus (Fabricius, 1775), is one of the most significant pests of oilseed rape. To shed light on past and current pollen beetle demography (dispersal, population size), 12 microsatellite markers were developed, and population genetic diversity and structure were analysed at different spatial scales in France and in Europe from 433 individuals collected in 18 winter oilseed rape fields. Genetic differentiation among the population samples was low but was significant between the Estonian sample and the rest of Europe. Isolation by distance was significant only at the European scale. Genetic variability was similar among the 18 population samples. Demographic inferences suggested a recent expansion of B. aeneus population size over Europe, possibly corresponding to an increase in oilseed rape crop area during past decades. Current population size and dispersal are not straightforward to estimate from the distribution of genetic variability in B. aeneus over Europe because of the complexity of the demographic history of this pest. Nevertheless, because gene flow was important enough to prevent strong genetic differentiation at large geographical scales, the management of pollen beetle populations should likely be thought of at a continental Europe level. © 2018 Society of Chemical Industry.

Overwintering of pollen beetles and their predators in oilseed rape and semi-natural habitats

Semi-natural habitats (SNH) maintain high levels of biodiversity and ecosystem functioning within agroecosystems. While management recommendations for SNH to promote biodiversity and optimize natural pest control, have mostly focused on improving food resources for predators, overwintering conditions have hitherto received less attention. The success of conservation biological control is often hampered by a lack of knowledge on how different habitats and their associated traits drive the overwintering of both, crop pests and their natural enemies. Moreover, there are concerns that SNH may act as reservoirs for crop pests.We investigated the overwintering of pollen beetles (Meligethes spp.) and their predators (predatory ground beetles) across 40 habitats of all major types of SNH as well as 8 winter oilseed rape (WOSR) fields, to identify the importance of type and key traits of habitats. Overwintering of pollen beetles was higher in WOSR fields (7 individuals per 0.25 m2) and forest edges (4) compared to flower strips (2) or forest interiors (1). Similarly, overwintering of predatory ground beetles was higher in forest edges and WOSR fields (11 and 10 individuals, respectively) than in flower strips, meadows or hedgerows (4, 4 and 2 individuals). The predator-prey ratio (number of predatory ground beetles / number of pollen beetles) was higher in forest edges than in flower strips, meadows or hedgerows, and intermediate in WOSR fields. Pollen beetle numbers were negatively associated with increasing litter cover and positively with increasing bare soil, while predatory ground beetles were positively associated with soil bulk density and bare soil cover. The proportion of SNH at the landscape level (1 km radius) did not affect overwintering pollen beetles, predatory ground beetles or predator-prey ratio.We conclude that WOSR fields, rather than SNH are the major overwintering habitat and source of spring colonisation of WOSR by pollen beetles. Forest edges promote predatory ground beetles rather than pollen beetle populations according to their disproportionally high value of overwintering individuals. Agri-environmental measures to promote predatory ground beetles rather than pollen beetles should promote overwintering habitats with compact soils and high litter cover.

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

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. 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. 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.

Susceptibility of Danish pollen beetle populations against λ-cyhalothrin and thiacloprid

Winter oilseed rape is an important crop in agriculture, where it plays an essential role in cereal-based crop rotations. The main pest in this crop is the pollen beetle which feeds on developing buds. Since 1999, pyrethroid resistance in the pollen beetle has spread throughout Europe. Denmark and Sweden are of interest due to the early detection of pyrethroid-resistant pollen beetle and because of the presence of both target-site and metabolic resistance in the populations. For almost ten years, neonicotinoid insecticides have been used in oilseed rape as well. In this study, the current resistance situation in Danish pollen beetle populations was conducted. The susceptibility of pollen beetle to the synthetic pyrethroid λ-cyhalothrin as well as the neonicotinoid thiacloprid was investigated. Sixty-eight Danish, as well as 6 Swedish and 10 German populations collected between 2014 and 2016 were tested with λ-cyhalothrin. Two-thirds of the Danish populations collected were classified as pyrethroid resistant, whereas Swedish populations were mostly susceptible and all German populations tested were resistant, mostly at higher levels than those detected in Danish strains. Toxicological parameters were calculated by a two-parameter log-logistic model and showed up to 58-fold variation at LC50 for λ-cyhalothrin and up to 17-fold variation at LC50 for thiacloprid. However, almost all Danish populations were susceptible against thiacloprid and the German populations showed up to sevenfold variation in thiacloprid bioassay. In conclusion, pyrethroid resistance is widespread in Danish pollen beetle populations with regional differences, whereas Danish populations are susceptible to thiacloprid in all regions.

Negative correlations between the susceptibilities of Czech and Slovak pollen beetle populations to lambda-cyhalothrin and chlorpyrifos-ethyl in 2014 and 2015

Sixty-five Czech Meligethes populations were tested against lambda-cyhalothrin and chlorpyrifos-ethyl in 2014. In 2015, totally sixty Meligethes populations, some of which sampled also in Slovakia, were tested against the two insecticides. Adult vial tests by IRAC (Insecticide Resistance Action Committee) were used for testing (No. 011 v. 3 for lambda-cyhalothrin and No. 025 for chlorpyrifos-ethyl). For each of the tested populations the LC<sub>50</sub>, LC<sub>90</sub>, and in 2015 also LC<sub>95</sub> values were determined for both these insecticides. Correlation analyses were made with transformed (log10 transformation) LC values. No significant correlation was recorded between the LC<sub>50</sub> values. Contrary to that, significant (P < 0.05) negative (r values for negative) correlations were recorded between the LC<sub>90</sub> and LC<sub>95</sub> values. Pyrethroid resistance in pollen beetle populations should indicate their slightly higher susceptibility to chlorpyrifos-ethyl.

A de novo transcriptome of European pollen beetle populations and its analysis, with special reference to insecticide action and resistance

The pollen beetle Meligethes aeneus is the most important coleopteran pest in European oilseed rape cultivation, annually infesting millions of hectares and responsible for substantial yield losses if not kept under economic damage thresholds. This species is primarily controlled with insecticides but has recently developed high levels of resistance to the pyrethroid class. The aim of the present study was to provide a transcriptomic resource to investigate mechanisms of resistance. cDNA was sequenced on both Roche (Indianapolis, IN, USA) and Illumina (LGC Genomics, Berlin, Germany) platforms, resulting in a total of ∼53 m reads which assembled into 43 396 expressed sequence tags (ESTs). Manual annotation revealed good coverage of genes encoding insecticide target sites and detoxification enzymes. A total of 77 nonredundant cytochrome P450 genes were identified. Mapping of Illumina RNAseq sequences (from susceptible and pyrethroid-resistant strains) against the reference transcriptome identified a cytochrome P450 (CYP6BQ23) as highly overexpressed in pyrethroid resistance strains. Single-nucleotide polymorphism analysis confirmed the presence of a target-site resistance mutation (L1014F) in the voltage-gated sodium channel of one resistant strain. Our results provide new insights into the important genes associated with pyrethroid resistance in M. aeneus. Furthermore, a comprehensive EST resource is provided for future studies on insecticide modes of action and resistance mechanisms in pollen beetle.

Baseline susceptibility and insecticide resistance monitoring in European populations of Meligethes aeneus and Ceutorhynchus assimilis collected in winter oilseed rape

AbstractPollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and cabbage seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), are important pests in the production of European winter oilseed rape, Brassica napus L. (Brassicaceae), which is grown on several million hectares in Europe. Insecticide treatments are common to control both pests once they exceed economic damage thresholds; however, not many chemical classes are available for their control in European oilseed rape. Particularly pollen beetles recently developed high levels of pyrethroid resistance impairing field control at recommended rates in many countries, whereas no resistance is yet reported to another important insecticide, thiacloprid. The major objective of this study was to investigate the spatio‐temporal susceptibility status of pollen beetle against the recently introduced insecticide thiacloprid. From 2009 to 2012 more than 630 populations of pollen beetle collected in 13 countries were monitored for resistance to thiacloprid by using an adult vial test. No shifting to lower susceptibility of pollen beetle to thiacloprid has been observed between 2009 and 2012. Furthermore, we were able to show that pollen beetle larvae are extremely susceptible to thiacloprid, whereas within strains larvae are significantly more resistant than adults to pyrethroids such as lambda‐cyhalothrin. Dose‐response data for thiacloprid against cabbage seed weevil populations collected in 2011 in Germany, Sweden, and Ukraine showed a 10‐fold higher intrinsic sensitivity compared to pollen beetle, and showed only a low variation in response. In addition, we also tested 17 cabbage seed weevil populations collected in five countries against lambda‐cyhalothrin with low variation in response (three‐fold), suggesting full baseline susceptibility and no resistance to pyrethroids. The implications of the data presented for resistance management in coleopteran pests in winter oilseed rape are discussed.

Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus

The pollen beetle (Meligethes aeneus F.) is widespread throughout much of Europe where it is a major coleopteran pest of oilseed rape (Brassica napus). The reliance on synthetic insecticides for control, particularly the pyrethroid class, has led to the development of populations with high levels of resistance. Resistance to pyrethroids is now widespread throughout Europe and is thought to be mediated by enhanced detoxification by cytochrome P450ś and/or mutation of the pyrethroid target-site, the voltage-gated sodium channel. However, in the case of cytochrome P450 mediated detoxification, the specific enzyme(s) involved has (have) not yet been identified. In this study a degenerate PCR approach was used to identify ten partial P450 gene sequences from pollen beetle. Quantitative PCR was then used to examine the level of expression of these genes in a range of pollen beetle populations that showed differing levels of resistance to pyrethroids in bioassays. The study revealed a single P450 gene, CYP6BQ23, which is significantly and highly overexpressed (up to ∼900-fold) in adults and larvae of pyrethroid resistant strains compared to susceptible strains. CYP6BQ23 overexpression is significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Functional recombinant expression of full length CYP6BQ23 along with cytochrome P450 reductase in an insect (Sf9) cell line showed that it is able to efficiently metabolise deltamethrin to 4-hydroxy deltamethrin. Furthermore we demonstrated by detection of 4-hydroxy tau-fluvalinate using ESI-TOF MS/MS that functionally expressed CYP6BQ23 also metabolizes tau-fluvalinate. A protein model was generated and subsequent docking simulations revealed the predicted substrate-binding mode of both deltamethrin and tau-fluvalinate to CYP6BQ23. Taken together these results strongly suggest that the overexpression of CYP6BQ23 is the primary mechanism conferring pyrethroid resistance in pollen beetle populations throughout much of Europe.

RNA‐sequencing analysis reveals abundant developmental stage‐specific and immunity‐related genes in the pollen beetle Meligethes aeneus

The pollen beetle (Meligethes aeneus) is a major pest of oilseed rape (Brassica napus) and other cruciferous crops in Europe. Pesticide-resistant pollen beetle populations are emerging, increasing the economic impact of this species. We isolated total RNA from the larval and adult stages, the latter either naïve or immunized by injection with bacteria and yeast. High-throughput RNA sequencing (RNA-Seq) was carried out to establish a comprehensive transcriptome catalogue and to screen for developmental stage-specific and immunity-related transcripts. We assembled the transcriptome de novo by combining sequence tags from all developmental stages and treatments. Gene expression data based on normalized read counts revealed several functional gene categories that were differentially expressed between larvae and adults, particularly genes associated with digestion and detoxification that were induced in larvae, and genes associated with reproduction and environmental signalling that were induced in adults. We also identified many genes associated with microbe recognition, immunity-related signalling and defence effectors, such as antimicrobial peptides (AMPs) and lysozymes. Digital gene expression analysis revealed significant differences in the profile of AMPs expressed in larvae, naïve adults and immune-challenged adults, providing insight into the steady-state differences between developmental stages and the complex transcriptional remodelling that occurs following the induction of immunity. Our data provide insight into the adaptive mechanisms used by phytophagous insects and could lead to the development of more effective control strategies for insect pests.

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.

Cytochrome P450 mediated pyrethroid resistance in European populations of Meligethes aeneus (Coleoptera: Nitidulidae)

Pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae) is a major pest on several million hectares in European winter oilseed rape cultivation. Synthetic pyrethroids have been successfully used for many years to keep them under economic damage thresholds. Recently wide-spread resistance development to pyrethroids in pollen beetle populations was described in many European countries, including Germany, France, Poland, Denmark and others. Resistance monitoring is conducted by incubating beetles for 24 h in glass vials coated with different concentrations of lambda-cyhalothrin. Using such an assay format we were able to show cross-resistance to other pyrethroids, such as deltamethrin, cypermethrin, and to a somewhat lower extent bifenthrin, etofenprox and tau-fluvalinate. Here we also investigated in more detail in 27 different populations the biochemical mechanism of pyrethroid resistance. Synergism experiments revealed a high synergistic potential for piperonyl butoxide in vivo, whereas other compounds such as S,S,S-tributylphosphorotrithioate and diethylmaleimide failed to suppress pyrethroid resistance. Incubating microsomal fractions of pollen beetle with deltamethrin and subsequent LC–MS/MS analysis revealed 4-OH-deltamethrin as the major metabolite. Metabolite formation in vitro and pyrethroid resistance in vivo is correlated and inhibition trials with piperonyl butoxide, tebuconazole and aminobenzotriazole suggest the involvement of cytochrome P450′s. Furthermore we were able to show cross-resistance to tau-fluvalinate which is supported by the competitive inhibition of 4-OH-deltamethrin formation by increasing concentrations of tau-fluvalinate in microsomal hydroxylation assays. Although we provided clear experimental evidence for an oxidative mechanism of resistance in numerous populations, other mechanisms might be involved based on the data discussed.

Pyrethroid resistance and thiacloprid baseline susceptibility of European populations of Meligethes aeneus (Coleoptera: Nitidulidae) collected in winter oilseed rape

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae), is a major pest in European winter oilseed rape. Recently, control failures with pyrethroid insecticides commonly used to control this pest have been reported in many European countries. For resistance management purposes, the neonicotinoid insecticide thiacloprid was widely introduced as a new mode of action for pollen beetle control. A number of pollen beetle populations collected in Germany, France, Austria, Great Britain, Sweden, Denmark, Finland, Poland, Czech Republic and Ukraine were tested for pyrethroid resistance using lambda-cyhalothrin-coated glass vials (adult vial test). Most of the populations tested exhibited substantial levels of resistance to lambda-cyhalothrin, and resistance ratios ranged from < 10 to > 2000. A similar resistance monitoring bioassay for the neonicotinoid insecticide thiacloprid was developed and validated by assessing baseline susceptibility data for 88 European pollen beetle populations. A variation of less than fivefold in response to thiacloprid was detected. The thiacloprid adult vial bioassay is based on glass vials coated with an oil-dispersion-based formulation of thiacloprid, resulting in a much better bioavailability compared with technical material. Analytical measurements revealed a > 56 and 28 day stability of thiacloprid and lambda-cyhalothrin in coated glass vials at room temperature, respectively. No cross-resistance between thiacloprid and lambda-cyhalothrin based on log-dose probit-mortality data was detected. Pyrethroid resistance in many European populations of M. aeneus was confirmed, whereas all populations are susceptible to thiacloprid when tested in a newly designed and validated monitoring bioassay based on glass vials coated with oil-dispersion-formulated thiacloprid. Based on the homogeneous results, it is concluded that thiacloprid could be an important chemical tool for pollen beetle resistance management strategies in European winter oilseed rape.

Pyrethroid resistance monitoring in European populations of pollen beetle (Meligethes spp.): a coordinated approach through the Insecticide Resistance Action Committee (IRAC)

Pollen beetle (Meligethes spp.) is a major pest of European oilseed rape crops. Its resistance to pyrethroid insecticides has been recorded in samples of beetles collected in Europe since at least 1999, and problems with the control of the beetle in the field have been widely reported. In 2007, a Pollen Beetle Working Group was formed through the Insecticide Resistance Action Committee (IRAC) in order to coordinate efforts for surveying pyrethroid resistance development. The results of the first 3 years of the pollen beetle pyrethroid susceptibility survey using a laboratory test are presented in this paper. Resistant beetle samples were collected from 20 of the 21 countries surveyed, with a general trend of increasing frequency and spread of resistant samples in European oilseed-rape-growing regions. Pyrethroid-resistant beetles dominate in Western and Central Europe and are becoming established in the North and East, the main oilseed-rape-growing areas of Europe. The development and spread of pyrethroid-resistant pollen beetles highlights the need for effective management strategies for oilseed rape insect pests.

Genetic diversity in European pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae), populations assessed using AFLP analysis

The pollen beetle (Meligethes aeneus) is one of the most important insect pests of oilseed rape (Brassica napus), and extensive use of insecticides is required to protect crop yields. To meet the challenges set by agricultural demands for more sustainable production and changing climate more information about pest biology and population genetics is needed. Using genomic Amplified Fragment Length Polymorphism (AFLP) analysis, DNA polymorphism was studied in 14 field populations of pollen beetles, collected during 2004 in six European countries (Denmark, France, Finland, Germany, Sweden, and UK). Using one primer combination 410 polymorphic DNA fragments were obtained based on analysis of single beetles. AFLP profiles were analysed with similarity measures using the Nei and Li coefficient and dendrograms generated. Dendrograms constructed from distance matrices revealed clustering by population origin and assignment analysis generally supported the genotype classification. Principal component analysis of the fourteen groups resulted in wide dispersion but also connections between some groups. Statistical analysis using AMOVA showed that the levels of genetic variation within populations explained most of the variation. Migrant analysis suggested a low level of gene flow between pollen beetle populations at different geographical locations indicating little long range dispersal of pollen beetles. However, a Mantel test found no correlation between genetic and geographical distance. Apparently genetic differentiation among populations has a complex background and may involve factors such as local adaptation and founder effects.