Browsing by Subject "Neonicotinoid"

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    Effects of chronic pesticide and pathogen exposure on honey bee (Apis mellifera L.) health at the colony level
    (2018) Odemer, Richard; Bessei, Werner
    During the last decade the increasing number of honey bee colony losses has become a major concern of beekeepers and scientists worldwide. Extensive research and cooperation projects have been established to unravel this phenomenon. Among parasites, pathogens and environmental factors, the use of agrochemicals, most notably the class of neonicotinoid insecticides, are suspected to be a key factor for this collapse. Current approaches not only focus on colony collapse but also on the weakening of honey bees by the exposure to sublethal concentrations of such pesticides. Recently, the EFSA temporarily banned three neonicotinoids including clothianidin, imidacloprid and thiamethoxam, for the use in crops attractive to pollinators. Thiacloprid however, likewise a neonicotinoid insecticide, is still tolerated for agricultural use because it is considered less toxic to bees. Nevertheless, some publications indicate sublethal effects of this agent leading to impairments of the colony. A general problem for the study of such sublethal effects is that they often are measurable in individual bees without eliciting clear impact at the colony level. In addition, such effects might only have a consequence in combination with other stressors like pathogens. This thesis presents two new methodical approaches combining the controlled application of stressors to individual bees with an evaluation of the effects under field realistic conditions of free flying colonies. In all approaches, the bees were treated with a combination of different pesticides and/or a combination of pesticides and a pathogen in order to evaluate synergistic interactions. As pathogen, Nosema ceranae, a novel intracellular gut parasite introduced from Asia, was used. This parasite is considered to contribute to “CCD”-like symptoms (“colony collapse disorder”), particularly in Spain. In Retschnig et al. (2015), observation hives at two study sites (Hohenheim and Bern) were used to clarify possible synergistic effects when honey bees are exposed to pesticides of two different substance classes (thiacloprid and tau-fluvalinate), both in combination with an infection of N. ceranae. Mortality, flight activity and social behaviour of individually marked and treated worker bees were monitored. At the Hohenheim site, no impact from any of the treatments could be confirmed except a slightly higher flight activity of the Nosema treated bees. At the Bern site however, the pesticide treatments elicited a significant reduction of worker bee lifespan, whereas the Nosema infection resulted in higher ratios of motionless periods. Importantly and in contrast to several laboratory studies, in neither of the two sites an interaction among the pesticides and the pathogen could be confirmed. The inconsistency of our results suggests that the effects of both, sublethal application of pesticides and infection with N. ceranae were rather weak and that interaction among them may have been overemphasized. To extend this first approach in small observation colonies, Odemer & Rosenkranz (2018) focused on performance parameters such as colony development and overwintering in honey bee colonies, using the same pesticides as in the observation hives. Here, neither the single exposure to thiacloprid or tau-fluvalinate nor their combination had negative effects on the colony performance. However, the chronic application of the tau-fluvalinate significantly reduced the infestation with Varroa mites. In Odemer et al. (2018), a neonicotinoid (clothianidin) with an extraordinary high toxicity to bees was applied alone and in combination with N. ceranae and N. apis. A novel approach was developed with individually marked bees that were infected after hatching with a certain number of Nosema spores and introduced into mini-hives. In order to simulate worst case field conditions, the pesticide was then applied chronically in sublethal concentrations over the whole lifespan of the bees. Again in contrast to previous laboratory studies, no effect of the clothianidin treatment on mortality or flight activity could be observed. However, the lifespan of Nosema infected bees was significantly reduced compared to non-infected bees, but in agreement with the observation hive experiment, the combination of pesticide and pathogen did not reveal any synergistic effect. The results of the three experiments of this thesis indicate that (i) individual honey bees are less impaired by neonicotinoids if kept within the social environment of the colony and that (ii) sublethal concentrations of neonicotinoids in the field are not the main driver for colony losses. These statements refer exclusively to the honey bee colony as a eusocial superorganism that obviously is more resilient to pesticide exposure through mechanisms of “social buffering”.
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    Freisetzung von Neonicotinoiden aus der Saatgutbeizung in Guttation von Kulturpflanzen und deren Auswirkungen auf Honigbienen Apis mellifera L. (Hymenoptera: Apidae)
    (2015) Reetz, Jana E.; Zebitz, Claus P. W.
    Seed coating with the systemic neonicotinoids clothianidin, imidacloprid, and thiamethoxam was considered environmental justifiable and no side effects on non-target organisms such as honey bees were considered during the registration process as seed coatings. In 2008, sowings with neonicotinoid-coated corn has caused severe damages on honey bee colonies in the upper Rhine Valley, Germany. As a consequence, the seed coating with neonicotinoids in maize and wheat was suspended in Germany in May 2008; since May 2013 there is a temporary ban of the three neonicotinoids by the EU Regulation No. 485/2013. The release of systemic active substances (a.s.) in guttation of seed-coated plants, e.g. winter oilseed rape (WOR, Brassica napus L.), represents a possible contamination source for non-target organisms and could actively be used as a water source by honey bees (Apis mellifera L.). The occurrence of guttation was examined and sampled under field conditions in maize, xtriticale and WOR. The residual analysis of guttation from seed-coated WOR revealed the release of residues up to 130 µg a.s. L-1 in autumn (Reetz et al. 2015). However, even the highest residues in WOR guttation are considerably lower than those in guttation of maize (up to 8,000 µg a.s. L-1) or xtriticale (up to 1,300 µg a.s. L-1; Reetz et al. 2011). In spring, the released residues in WOR guttation are lower than in early autumn (~30 µg a.s. L-1) and continue to decline steadily until flowering. Considerable high amounts of residues have been released by maize (spring crop) over a long period of the crop cycle. Laboratory investigations (according to OECD-Guideline 213) showed that feeding of isolated honey bees with a sugar/guttation-solution from seed-coated WOR leads to a mortality less than 20 % (Wallner et al. 2012), but this way of exposure is not similar to the situation of water foraging honey bees. Observations of water foraging honey bees in the field are nearly impossible. Therefore, honey-sac content of foragers returning to the hive were analysed for residues (Reetz and Wallner 2014). These experiments showed that on the one side the weight of honey sacs is lower during autumn at the same time when high residues in guttation of seed-coated WOR occur than in summer, and on the other side, that the intake of water is increased by the factor of 25 compared to the amount of nectar, which seems to be associated with the absence of nectar sources during autumn (Reetz et al. 2012). There seems to be no exclusive season- or daytime-depending water collecting activity in honey bee colonies in temperate zones. Therefore, the collection of guttation from seed-coated plants by foraging honey bees is likely. However, during summer and the periods of high nectar flows honey bees might gather rather runny nectar as a replacement for water than WOR guttation. Honey bees gathering on WOR guttation were just occasionally observed in a small-patterned landscape, but more frequently in the field site with intensive agriculture and a reduced variety of alternative water sources. HPLC-MS-analysis of honey sacs (n= 204) reveal that residues of thiamethoxam are detectable in 19 % (n= 38) of the honey-sac contents with a range of 0.3 to 0.95 µg a.s. per litre (LOQ= 0.3 µg a.s. L-1; Reetz et al. 2015 accepted). In 12 % (n= 24) of the samples, thiamethoxam could be detected in concentrations below LOQ. Clothianidin and its metabolite TZMU were measured in one sample each (0.5 %) at concentrations below LOQ (clothianidin) and LOD (TZMU), respectively. Based on these experiments, it has been proven that honey bees use guttation of seed-coated WOR as water source in absence of alternative nearby water sources. Thus, during a short period of about a few weeks in autumn, when the highest residues are released in WOR guttation, there might theoretically be a risk for single honey bees. Guttation of xtriticale and WOR is just temporary present in the field, whereas guttation of maize is present in the leaf sheaths during the day due to the funnel function of the maize leaves. Additionally to theses facts, there is a low water demand in honey bee colonies during autumn in contrast to the occurrence of maize guttation, which occurs at the same time when honey bee colonies raise and have an increasing demand of water. The current evaluation of short-term effects of chronic exposure to sublethal concentrations of neonicotinoids in pollen on honey bees at colony level is based on the application of higher concentrations (2 ppb clothianidin; Sandrock et al. 2014) than detected in the honey-sac contents of the water foraging honey bees in this experiment (< 1 µg a.s. L-1; < 1 ppb). Based on these threshold values for side effects by chronic feeding of neonicotinoids, the concentrations of residues measured in the honey sacs of water foraging honey bees seem to have still less potential for side effects on single honey bees or on colony level.
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    Monitoring, mechanisms and management of insecticide resistance and insecticide mode of action in coleopteran pests of winter oilseed rape with special reference to neonicotinoid insecticides under laboratory and applied aspects
    (2014) Zimmer, Christoph Thomas; Zebitz, Claus P. W.
    Winter oilseed rape, Brassica napus L., has become a vital part of cereal-based crop rotations in Europe. It is attacked by numerous insect pests and their control relies on the intensive use of insecticides (compared to other broad acre crops). The exclusive and continuous use of pyrethroid insecticides for almost twenty years led to an enormous selection pressure and facilitated the development of resistance in oilseed rape pests in Europe. Unsurprising three out of the five major pests of the order Coleoptera are reported to be pyrethroid resistant at present: the pollen beetle, Meligethes aeneus F.; the cabbage stem flea beetle, Psylliodes chrysocephala L. and the cabbage seed weevil, Ceutorhynchus assimilis PAYK.. An adult vial bioassay, which is based on insecticide coated glass vials, was used to monitor the spread and strength of pyrethroid resistance and to determine cross-resistance pattern in pollen beetle and cabbage stem flea beetle. Furthermore, baseline susceptibility towards lambda-cyhalothrin (a widely used pyrethroid) was also established for the cabbage seed weevil. The vial bioassay methodology was adapted to thiacloprid, a neonicotinoid insecticide, to determine baseline susceptibility and to provide a methodology to allow long-term susceptibility monitoring of pollen beetle and cabbage seed weevil. Thiacloprid monitoring revealed that pollen beetle and cabbage seed weevil populations collected across Europe in 2009-2012 and 2012 respectively were highly susceptible to this insecticide class. Metabolism studies using native microsomal preparations as the enzyme source and deltamethrin as substrate revealed metabolism of deltamethrin with 4-OH-deltamethrin being the major metabolite. Metabolite formation in vitro was correlated with the observed pyrethroid resistance level in vivo and was suppressible by PBO. A degenerate PCR approach was used to identify partial P450 gene sequences from pollen beetle. qRT-PCR screening covering a range of pollen beetle populations differing in levels of pyrethroid resistance identified a single P450, CYP6BQ23, as significantly and highly overexpressed (up to ~900-fold) in resistant strains compared to susceptible strains. The expression of CYP6BQ23 was significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Recombinant expression of this P450 in an insect cell line demonstrated that it is capable of hydroxylating deltamethrin and tau-fluvalinate. The turnover of these pyrethroids by CYP6BQ23 is in line with the observed moderate cross-resistant phenotype. Molecular modeling suggested a better fit of deltamethrin into the active site of CYP6BQ23 compared to tau-fluvalinate also supporting the biochemical results. The occurrence of target-site resistance was investigated by single nucleotide polymorphism (SNP) analysis of the para-locus encoding the voltage-gated sodium channel (VGSC) in insects. To achieve this goal a partial fragment (domain IIS4-6) encoding an important region of the pyrethroid binding site was PCR amplified and screened for non-synonymous SNPs. One SNP was identified causing a leucine to phenylalanine substitution at amino acid residue number 1014 (Musca domestica L. numbering), well known as knock down resistance (kdr) conferring an absolute cross-resistance to pyrethroids and DDT in various insect species. Sequencing of the very same gene region in the cabbage stem flea beetle also revealed the presence of the L1014F kdr mutation in pyrethroid resistant flea beetle populations, thus explaining the strong cross-resistance pattern observed in vitro. Most mechanistic studies of resistance have focused on elucidating the contribution of particular genes/gene families to pyrethroid resistance. To generate a comprehensive sequence resource and to elucidate global changes in gene regulation related to insecticide resistance in pollen beetle a de novo transcriptome was assembled from sequence pools generated by next-generation sequencing. RNA-sequencing of three pyrethroid resistant and one highly susceptible reference population allowed a global gene expression analysis by short read mapping against the generated transcriptome, as well as a SNP analysis. The implications of these results for resistance management in coleopteran pests in winter oilseed rape and opportunities for future work are discussed.