Towards Integrated Pest and Pollinator Management in Intensive Pear Cultivation: A Case Study from Belgium.

Recently, the concept of Integrated Pest Management (IPM) was further extended into Integrated Pest and Pollinator Management (IPPM). Implementation of IPPM strategies entails the combination of actions for pest and pollinator management providing complementary or synergistic benefits for yield and/or quality of the harvest. The aim of this study was to examine IPPM elements (i.e., mixed hedgerow, nesting boxes for mason bees, Osmia spp.) and demonstrate their impact in the practical context of modern commercial fruit cultivation in a 4-year case study in an intensive 'Conference' pear orchard. The outcomes of visual observations during transect walks and molecular analysis of pollen collected by mason bees, showed the importance of additional floral resources for the presence of mason bees and other pollinating insects in the orchard environment. Pear quality assessments indicated that insect-mediated pollination had a significant positive impact, with a tendency for higher quality pears in the close vicinity of Osmia nesting boxes. However, despite the fact that pear pollen was also detected in Osmia spp. nest cells, the amount and frequency of pear pollen collection for their nest built-up turned out to be rather low. In the same intensive pear orchard studied for pollination effects, we simultaneously demonstrate the impact of a mixed hedgerow to enhance integrated pest control.

Commercial bumblebee hives to assess an anthropogenic environment for pollinator support: a case study in the region of Ghent (Belgium).

Anthropogenic changes of the environment influence the distribution and abundance of pollinators such as bumblebees and have been proposed as one of the main causes in their worldwide decline. In order to evaluate the impact of expanding anthropogenic landscapes on supporting pollinator potential, reliable tools are needed. Bombus terrestris is one of the most abundant bumblebee species in Europe, and these bumblebees are known as generalist pollinators of not only wild flowers in nature but also of crops in agriculture. For more than two decades, these bumblebees have been commercially mass reared for biological pollination in greenhouses. In this project, we placed commercial hives of the bumblebee B. terrestris containing one queen and 40 workers, in three different locations in the region of Ghent (Belgium), and the performance of these hives was followed during a 4-week period in spring 2012. In parallel, we determined the floral richness and diversity index in the chosen study sites. The sites consisted of a rich urban environment with patchy green areas opposed to an urban environment with poor landscape metrics; a third rural study site showed average positive landscape metrics. The results demonstrated that the hive biomass and numbers of workers increased significantly in the rich compared to the poor environment, providing a mechanism to discriminate between study sites. In addition, the bumblebee-collected pollen showed that the flowering plants Salix spp. and Rosaceae/Prunus spp. are dominant food sources in all anthropogenic environments during early spring. Finally, the results are discussed in relation to the optimization of the experimental setup and to the use of commercial bumblebee hives in assessing local pollinator support within any given environment.

Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects.

Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

Assessment of gustatory responses to different sugars in harnessed and free-moving bumblebee workers (Bombus terrestris).

For bumblebee colony survival, sugar responses are crucial as nectar is the main carbohydrate source and flower choice is likely determined by sugar composition. This study used a bioassay both with harnessed and with free-moving workers of the bumblebee Bombus terrestris to study the gustatory response to the 3 major plant sugars by both groups. In harnessed workers of B. terrestris, a concentration of 5.5% of fructose and glucose was required to induce the proboscis extension reflex in 50% of the workers, whereas for sucrose, a much higher concentration of 40% was needed. In contrast, free-moving workers given a choice between 30% glucose, 30% sucrose, 30% fructose, and water showed a strong preference for sucrose (66% of individuals) compared with 18% for glucose and 16% for fructose; water was never chosen. Familiarization with 30% fructose provoked a significant increase in preference toward fructose, indicating plasticity. In addition, by amputation of the tarsi, it was found that tarsi plays a role in the sugar response with especially the foreleg tarsi being involved in the response to fructose. Our results demonstrated that sugar response is different in free-moving versus harnessed bumblebee workers and that tarsi plays a role in sugar perception.

Dietary chlorantraniliprole suppresses reproduction in worker bumblebees.

BACKGROUND: Pollinators such as the bumblebee, Bombus terrestris, fulfil a crucial role in agriculture. In this context, tests were conducted with the insecticide chlorantraniliprole (Coragen®) as a model compound active on the ryanodine receptor of insects. RESULTS: Chronic oral exposure via pollen induced lethargic behaviour in B. terrestris workers and their offspring (drones). Indeed, in nests exposed to 0.4 mg L(-1) , representing 1/100 of the concentration recommended for use in the field, workers and drones did not take their defensive position upon stimulation and they were less active than non-exposed insects. The different risk assessment tests used here demonstrated that contact and pollen exposure had no effect on bumblebee worker survival, whereas oral exposure via sugar water caused both acute (72 h LC50 = 13 mg L(-1) ) and chronic (7 week LC50 = 7 mg L(-1) ) toxicity. Severe sublethal effects on reproduction were recorded in nests orally exposed to pollen treated with chlorantraniliprole. CONCLUSION: The present study identified an important physiological endpoint of sublethal effects on reproduction, as this is associated with lethargic behaviour after oral intake. As such, this is a factor that should now be incorporated into future risk assessments. Secondly, it confirmed that the assessment of sublethal effects on behaviour is needed for adequate risk assessment of 'potentially deleterious' compounds with a neurogenic target, as is also pointed out in the recent European Food Safety Authority (EFSA) guidelines.

Safety and acquisition potential of Metarhizium anisopliae in entomovectoring with bumble bees, Bombus terrestris.

In the context of integrated pest management with biological control and reduced pesticide use, dissemination of entomopathogenic fungi with insects has the potency to protect crops and specifically their flowers against pests and diseases. But before implementation of such entomovectoring system, a measurement of risks of the microbial biocontrol agent toward the vectoring insect is crucial. The essential contributions of this project are that 1) exposure of bumble bees, Bombus terrestris (L.) to powder containing 10(7) spores of the commercial biocontrol agent Metarhizium anisopliae strain F52 (Biol020) per gram, was safe; and 2) that when bumble bees had walked through this spore concentration (10(7) spores per gram) in a dispenser, their body carried 9.3 +/- 1 x 10(6) spores/bumble bee, and this was still 2.6 10(6) spores after a flight of 60 s, representing the average time to fly from the dispenser to the crop flowers. 3) In contrast, a 100-fold higher spore concentration (10(9) spores per gram powder) was highly toxic and the acquisition on the bumble bee body was only 2.5 times higher. Based on these data, future studies can start investigating the protection efficacy of this entomovector system with M. anisopliae and bumble bees without harming the vector and with a loading of the vector considered enough to obtain a good inoculation into and protection of the flowers.

The non-target impact of spinosyns on beneficial arthropods.

Spinosyn-based products, mostly spinosad, have been widely recommended by extension specialists and agribusiness companies; consequently, they have been used to control various pests in many different cropping systems. Following the worldwide adoption of spinosad-based products for integrated and organic farming, an increasing number of ecotoxicological studies have been published in the past 10 years. These studies are primarily related to the risk assessment of spinosad towards beneficial arthropods. This review takes into account recent data with the aim of (i) highlighting potentially adverse effects of spinosyns on beneficial arthropods (and hence on ecosystem services that they provide in agroecosystems), (ii) clarifying the range of methods used to address spinosyn side effects on biocontrol agents and pollinators in order to provide new insights for the development of more accurate bioassays, (iii) identifying pitfalls when analysing laboratory results to assess field risks and (iv) gaining increasing knowledge on side effects when using spinosad for integrated pest management (IPM) programmes and organic farming. For the first time, a thorough review of possible risks of spinosad and novel spinosyns (such as spinetoram) to beneficial arthropods (notably natural enemies and pollinators) is provided. The acute lethal effect and multiple sublethal effects have been identified in almost all arthropod groups studied. This review will help to optimise the future use of spinosad and new spinosyns in IPM programmes and for organic farming, notably by preventing the possible side effects of spinosyns on beneficial arthropods.

Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment.

Neonicotinoid insecticides are successfully applied to control pests in a variety of agricultural crops; however, they may not only affect pest insects but also non-target organisms such as pollinators. This review summarizes, for the first time, 15 years of research on the hazards of neonicotinoids to bees including honey bees, bumble bees and solitary bees. The focus of the paper is on three different key aspects determining the risks of neonicotinoid field concentrations for bee populations: (1) the environmental neonicotinoid residue levels in plants, bees and bee products in relation to pesticide application, (2) the reported side-effects with special attention for sublethal effects, and (3) the usefulness for the evaluation of neonicotinoids of an already existing risk assessment scheme for systemic compounds. Although environmental residue levels of neonicotinoids were found to be lower than acute/chronic toxicity levels, there is still a lack of reliable data as most analyses were conducted near the detection limit and for only few crops. Many laboratory studies described lethal and sublethal effects of neonicotinoids on the foraging behavior, and learning and memory abilities of bees, while no effects were observed in field studies at field-realistic dosages. The proposed risk assessment scheme for systemic compounds was shown to be applicable to assess the risk for side-effects of neonicotinoids as it considers the effect on different life stages and different levels of biological organization (organism versus colony). Future research studies should be conducted with field-realistic concentrations, relevant exposure and evaluation durations. Molecular markers may be used to improve risk assessment by a better understanding of the mode of action (interaction with receptors) of neonicotinoids in bees leading to the identification of environmentally safer compounds.

Miniature-dispenser-based bioassay to evaluate the compatibility of powder formulations used in an entomovectoring approach.

BACKGROUND: Entomovectoring as a plant protection strategy demands the design of an appropriate bioassay to assess the risks of potential side effects of the powder formulations in the dispenser towards the vectoring insect. This study reports on the development of a laboratory miniature-dispenser-based bioassay. This bioassay system was used to investigate the compatibility of five model products, Prestop-Mix, Signum, kaolin, wheat flour and cellulose, with the bumblebee, Bombus terrestris L. RESULTS: The laboratory one-way miniature-dispenser bioassay showed that the fungicides and the carrier/diluent kaolin caused a worker mortality of > 70% after 5 weeks of exposure, while worker loss with wheat flour and cellulose was no higher than in the blank control (i.e. empty miniature dispenser) (<25%). The laboratory two-way miniature-dispenser bioassay comprised separated passageways and demonstrated that only kaolin was toxic (89 ± 11%). These results were also confirmed in a flight-cage experiment. In addition, a negative effect was observed against reproduction/colony development when nests were exposed to kaolin (P < 0.05) in the two-way miniature-dispenser and flight-cage bioassays. CONCLUSIONS: In the context of entomovectoring technology, the developed laboratory two-way miniature-dispenser bioassay gives a reliable prediction of the hazards associated with powder products. Additionally, the present data indicate the possibility of using cellulose and kaolin as respective negative and positive control carriers/diluents in future risk assessment experiments. Overall, the results show that, apart from kaolin, the tested fungicides and carriers/diluents are safe to be used with B. terrestris.

Assessment of side-effects by Ludox TMA silica nanoparticles following a dietary exposure on the bumblebee Bombus terrestris.

We assessed lethal and sublethal side-effects of Ludox TMA silica nanoparticles on a terrestrial pollinator, Bombus terrestris (Linnaeus), via a dietary exposure. Dynamic light scattering analysis confirmed that silica Ludox TMA nanoparticles remained in suspension in the drinking sugar water. Exposure of bumblebee microcolonies during 7 weeks to the different nanoparticle concentrations (high: 34, 170 and 340 mg/l and low: 34 and 340 µg/l) did not cause worker mortality compared to the controls. Also no effect on the worker foraging behavior was observed after exposure to nanoparticles concentrations up to 340 µg/l. In contrast, the high concentrations (≥34 mg/l) resulted in a total loss of reproduction. Using histological analysis we confirmed severe midgut epithelial injury in intoxicated workers (≥34 mg/l). Despite the fact that these concentrations are much higher than the predicted environmental concentrations, precaution is still needed as information regarding their fate in the terrestrial environment and their potency to bioaccumulate and biomagnificate is lacking.

Lethal and sublethal side-effect assessment supports a more benign profile of spinetoram compared with spinosad in the bumblebee Bombus terrestris.

BACKGROUND: This study was undertaken to identify the potential side effects of the novel naturalyte insecticide spinetoram in comparison with spinosad on the bumblebee Bombus terrestris L. The potential lethal effects together with the ecologically relevant sublethal effects on aspects of bumblebee reproduction and foraging behaviour were evaluated. Bumblebee workers were exposed via direct contact with wet and dry residues under laboratory conditions to spinetoram at different concentrations, starting from the maximum field recommended concentration (MFRC) and then different dilutions (1/10, 1/100, 1/1000 and 1/10 000 of the MFRC), and compared with spinosad. In addition, the side effects via oral exposure in supplemented sugar water were assessed. RESULTS: Direct contact of B. terrestris workers with wet residues of spinosad and spinetoram showed spinetoram to be approximately 52 times less toxic than spinosad, while exposure to dry residues of spinetoram was about 8 times less toxic than exposure to those of spinosad. Oral treatment for 72 h (acute) indicated that spinetoram is about 4 times less toxic to B. terrestris workers compared with spinosad, while exposure for a longer period (i.e. 11 weeks) showed spinetoram to be 24 times less toxic. In addition, oral exposure to the two spinosyns resulted in detrimental sublethal effects on bumblebee reproduction. The no observed effect concentration (NOEC) for spinosad was 1/1000 of the MFRC, and 1/100 of the MFRC for spinetoram. Comparison between the chronic exposure bioassays assessing the sublethal effects on nest reproduction, with and without allowing for foraging behaviour, showed that the respective NOEC values for spinosad and spinetoram were similar over the two bioassays, indicating that there were no adverse effects by either spinosyn on the foraging of B. terrestris workers. CONCLUSION: Overall, the present results indicate that the use of spinetoram is safer for bumblebees by direct contact and oral exposure than the use of spinosad, and therefore it can be applied safely in combination with B. terrestris. Another important conclusion is that the present data provide strong evidence that neither spinosyn has a negative effect on the foraging behaviour of these beneficial insects. However, before drawing final conclusions, spinetoram and spinosad should also be evaluated in more realistic field-related situations for the assessment of potentially deleterious effects on foraging behaviour with the use of queenright colonies of B. terrestris.

Bombus terrestris as pollinator-and-vector to suppress Botrytis cinerea in greenhouse strawberry.

BACKGROUND: Bombus terrestris L. bumblebees are widely used as commercial pollinators, but they might also be of help in the battle against economically important crop diseases. This alternative control strategy is referred to as pollinator-and-vector technology. The present study was designed to investigate the capacity of B. terrestris to fulfil this role in greenhouse strawberry flowers, which were manually inoculated with a major plant pathogen, the grey mould Botrytis cinerea Pers.: Fr. A model microbiological control agent (MCA) product Prestop-Mix was loaded in a newly developed two-way bumblebee dispenser, and, in addition, the use of the diluent Maizena-Plus (corn starch) was tested. RESULTS: Importantly, loading of the MCA caused no adverse effects on bumblebee workers, with no loss of survival or impairment of flight activity of the workers during the 4 week flowering period. Secondly, vectoring of Prestop-Mix by bumblebees resulted in a higher crop production, as 71% of the flowers developed into healthy red strawberries at picking (preharvest yield) as compared with 54% in the controls. In addition, these strawberries were better protected, as 79% of the picked berries remained free of B. cinerea after a 2 day incubation (post-harvest yield), while this percentage was only 43% in the control. Overall, the total yield (preharvest × post-harvest) was 2-2.5 times higher than the total yield in the controls (24%) in plants exposed to bumblebees vectoring Prestop-Mix. Thirdly, the addition of the diluent Maizena-Plus to Prestop-Mix at 1:1 (w/w) resulted in a similar yield to that of Prestop-Mix used alone, and in no negative effects on the bumblebees, flowers and berries. CONCLUSIONS: This greenhouse study provides strong evidence that B. terrestris bumblebees can vector a MCA to reduce B. cinerea incidence in greenhouse strawberries, resulting in higher yields. Similar yields obtained in the treatments with Prestop-Mix and Prestop-Mix + Maizena-Plus suggest an equally efficient dissemination of the biocontrol agent into the flowers with only half the initial concentration of Prestop-Mix, which illustrates the importance of the diluent.

Impact of a perfluorinated organic compound PFOS on the terrestrial pollinator Bombus terrestris (Insecta, Hymenoptera).

Perfluorinated organic chemicals like perfluorooctane sulfonic acid (PFOS) are persistent environmental pollutants that have been measured in a great diversity of wildlife worldwide, especially in the aquatic compartment. However, little information is available on the presence and effects of PFOS in the terrestrial compartment. Therefore, we investigated in this project the risks for effects, bioaccumulation and potential mechanisms of activity of PFOS in the bumblebee Bombus terrestris L. (Hymenoptera: Apidae) that is an important worldwide pollinator in the terrestrial compartment of wildflowers and cultivated crops. The exposure to PFOS occurred orally via the drinking of treated sugar water in a wide range from 1 µg/l up to 10 mg/l, containing environmentally relevant as well as high concentrations, and this was done with use of microcolonies of B. terrestris in the laboratory. A chronic toxicity assay demonstrated high bumblebee worker mortality (up to 100%) with an LC(50) of 1.01 mg/l (R(2) = 0.98). In addition, PFOS posed strong detrimental reproductive effects, and these concerted with a dramatic reduction in ovarian size. HPLC-MS demonstrated a bioaccumulation factor of 27.9 for PFOS in bumblebee workers fed with sugar water containing 100 µg/l PFOS during 5 weeks (2184 ± 365 ng/g BW). Finally, potential mechanisms of activity were investigated to explain the significant impact of PFOS on survival and reproduction capacity of B. terrestris. Exposure of bumblebee workers to PFOS resulted in a significant decrease in mitochondrial electron transport activity (p = 0.035) and lipid amounts (p = 0.019), while the respective p-values were 0.58 and 0.12 for protein and glucose amounts. Hence, addition of PFOS to ecdysteroid responsive Drosophila melanogaster S2 cells resulted in a strong antagonistic action on the EcR-b.act.luc reporter construct, demonstrating that PFOS may exert its effects partially through an endocrine disrupting action via the insect molting hormone or ecdysteroid receptor.

Age- and task-dependent foraging gene expression in the bumblebee Bombus terrestris.

In eusocial insects, the division of labor within a colony, based on either age or size, is correlated with a differential foraging (for) gene expression and PKG activity. This article presents in the first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the open reading frame allowed phylogenetic tracing, which showed conservation of PKGs among social insects. Our results confirm the proposed role for PKGs in division of labor. Btfor gene expression is significantly higher in the larger foragers compared with the smaller sized nurses. More importantly, we discovered an age-related decrease in Btfor expression in both nursing and foraging bumblebees. We therefore speculate that the presence of BtFOR is required for correct adaptation to new external stimuli and rapid learning for foraging. In a second series of experiments, worker bumblebees of B. terrestris were treated with two insecticides imidacloprid and kinoprene, which have shown to cause impaired foraging behavior. Compared with controls, only the latter treatment resulted in a decreased Btfor expression, which concurs with a stimulation of ovarian growth and a shift in labor toward nest-related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides.

Development of a new dispenser for microbiological control agents and evaluation of dissemination by bumblebees in greenhouse strawberries.

BACKGROUND: To date, in modern agriculture, biological control strategies are increasingly becoming the preferred pest management approach. However, the success of microbiological control agents (MCAs) largely depends on efficient dissemination into the crop. The pollinator-and-vector technology employs pollinating insects like bees for a better dissemination. In this study, a new dispenser for bumblebee workers of Bombus terrestris L. was developed. Binab-T-vector and Prestop-Mix were used as two typical MCA products for dissemination. RESULTS: In a first series of experiments in the laboratory for optimisation, the newly developed dispenser was a two-way type dispenser, 20 cm long, with two rectangular compartments and different entrance and exit holes. In addition, the amounts of MCA loaded on the workers were 10 times higher with the new dispenser as compared with the side-by-side passageway (SSP) dispenser. Typically, the highest amounts were recovered from the thorax and legs of the workers. In a second series of experiments under greenhouse conditions with the use of queen-right B. terrestris hives, successful dissemination in strawberry flowers was obtained at different distances from the hive (0-8 m, 8-18 m and 18-21 m), and the workers inoculated the first, second and third flowers that were consecutively visited. In addition, the new dispenser caused no adverse effects on worker foraging intensity, whereas a dramatic reduction was scored with an SSP dispenser. Finally, the data suggested that it is necessary to refill the newly developed dispenser at 3 day intervals. CONCLUSIONS: The results demonstrated that, with the use of the newly developed dispenser, bumblebee workers carried high amounts of MCA, and this resulted in a successful dissemination of MCA into strawberry flowers.

Compatibility of traditional and novel acaricides with bumblebees (Bombus terrestris): a first laboratory assessment of toxicity and sublethal effects.

BACKGROUND: This project assessed the potential hazards of different classical and novel acaricides against an important non-target and beneficial insect for the pollination of wild flowers and cultivated crops, the bumblebee Bombus terrestris (L). Twenty-three acaricides used commercially in the control of phytophagous mites (Acari) were tested in greenhouses and/or the open field. Side effects included acute mortality and also sublethal effects on nest reproduction. The different compounds were administered in the laboratory via three different worst-case field scenario routes of exposure: dermal contact and orally via the drinking of treated sugar water and via treated pollen. The compounds were tested at their respective maximum field recommended concentration (MFRC), and, when strong lethal effects were observed, a dose-response assay with a dilution series of the MFRC was undertaken to calculate LC(50) values. RESULTS: From the different acaricide classes, several chemistries caused high levels of acute toxicity in bumblebee workers, especially bifenthrin and abamectin which resulted in 100% mortality by contact. In addition, several acaricides tested were found to have a detrimental effect on drone production. For oral exposures via treated sugar water, the dose-response assay showed the LC(50) values for abamectin, bifenazate, bifenthrin and etoxazole to be 1/15 MFRC (1.17 mg AI L(-1)), 1/10 MFRC (9.6 mg AI L(-1)), 1/83 MFRC (0.36 mg AI L(-1)) and 1/13 MFRC (4.4 mg AI L(-1)) respectively, indicating that their use should be carefully evaluated. CONCLUSION: Overall, the results suggest that most of the acaricides tested are compatible with bumblebees, with the exceptions of abamectin, bifenazate, bifenthrin and etoxazole. However, the risks also depended on the type of treatment. As a result, the sugar water treatment seems to present the worst-case situation of exposure, indicating that this approach is suitable for determining the hazards of pesticides against bumblebees. Finally, it is suggested that future tier testing under more field-related conditions is required for a final decision of their risks.

Risk assessment for side-effects of neonicotinoids against bumblebees with and without impairing foraging behavior.

Bombus terrestris bumblebees are important pollinators of wild flowers, and in modern agriculture they are used to guarantee pollination of vegetables and fruits. In the field it is likely that worker bees are exposed to pesticides during foraging. To date, several tests exist to assess lethal and sublethal side-effects of pesticides on bee survival, growth/development and reproduction. Within the context of ecotoxicology and insect physiology, we report the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behavior under laboratory conditions. In brief, the experimental setup of this behavior test consists of two artificial nests connected with a tube of about 20 cm and use of queenless micro-colonies of 5 workers. In one nest the worker bees constructed brood, and in the other food (sugar and pollen) was provided. Before exposure, the worker bees were allowed a training to forage for untreated food; afterwards this was replaced by treated food. Using this setup we investigated the effects of sublethal concentrations of the neonicotinoid insecticide imidacloprid, known to negatively affect the foraging behavior of bees. For comparison within the family of neonicotinoid insecticides, we also tested different concentrations of two other neonicotinoids: thiamethoxam and thiacloprid, in the laboratory with the new bioassay. Finally to evaluate the new bioassay, we also tested sublethal concentrations of imidacloprid in the greenhouse with use of queenright colonies of B. terrestris, and here worker bees needed to forage/fly for food that was placed at a distance of 3 m from their hives. In general, the experiments showed that concentrations that may be considered safe for bumblebees can have a negative influence on their foraging behavior. Therefore it is recommended that behavior tests should be included in risk assessment tests for highly toxic pesticides because impairment of the foraging behavior can result in a decreased pollination, lower reproduction and finally in colony mortality due to a lack of food.

Impact of Bacillus thuringiensis strains on survival, reproduction and foraging behaviour in bumblebees (Bombus terrestris).

BACKGROUND: Bacillus thuringiensis (Bt) and its protein crystals are used worldwide, either as a spray or when expressed in transgenic crops, for the control of pest insects. However, owing to their intensive use, there exists a debate regarding the involvement of this microbial insecticide in bee colony losses. In this study, in a tiered approach using laboratory microcolonies, an evaluation was made of the potential lethal and sublethal hazards on colony reproduction and foraging behaviour of workers of the bumblebee Bombus terrestris (L.) of two commercial Bt strains: kurstaki (Dipel) and aizawai (Xentari). Bumblebees, like honey bees, are intensively used in modern agriculture for pollination and fulfil a crucial role in the natural ecosystem. RESULTS: Exposure of bumblebees dermally or via treated pollen to either of the two Bt formulations at their field recommended rates (0.1%) caused no reduction in survival. However, when applied in the feeding sugar water, aizawai killed all workers at a concentration of 0.1%, but this lethal effect was lost at 0.01%. With respect to reproductive effects, kurstaki was harmless, while aizawai at 0.1% delivered in the feeding sugar water and pollen reduced reproduction by 100 and 31% respectively. Lower doses of 0.01% aizawai in the sugar water showed no more effect. In addition, kurstaki at 0.1% and aizawai at 0.01% in the feeding sugar water did not impair the foraging behaviour, resulting in normal nest colony performance. CONCLUSION: The results with kurstaki and aizawai demonstrated that, in general, the Bt strains are safe to B. terrestris bumblebees, although in some cases there were detrimental effects that depended on strain and route of exposure. In addition, the authors believe that to draw firm conclusions regarding the hazards of Bt to bumblebees would require more information on relevant concentrations of Bt products in the environment. Hence, routine testing for lethal and sublethal effects is recommended to ascertain combined use of Bt products and bumblebees in modern agriculture.

A laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris.

BACKGROUND: This study was undertaken to identify any potential adverse side effects of the use of seven microbiological control agents (MCAs) on the bumblebee, Bombus terrestris L., in the context of combined use in integrated pest management (IPM). AQ10 (Ampelomyces quisqualis), Binab-T-vector (Hypocrea parapilulifera + T. atroviride; 1/1), Prestop-Mix (Gliocladium catenulatum J1446), Serenade (Bacillus subtilis QST713), Trianum-P (Trichoderma harzianum T22), Botanigard (Beauveria bassiana GHA) and Granupom (Cydia pomonella granulovirus), comprising five biofungicides and two bioinsecticides, were investigated. Bumblebee workers were exposed under laboratory conditions to each MCA at its maximum field recommended concentration (MFRC) via three different routes of exposure: dermal contact and orally via either treated sugar water or pollen. RESULTS: The tested MCAs were found to be safe for workers of B. terrestris, with the exception of Botanigard and Serenade. Exposure to Botanigard via contact at its MFRC caused 92% mortality after 11 weeks, while the 1/10 MFRC killed 46% of exposed workers. For Serenade, topical contact and oral delivery via sugar water resulted in 88 and 100% worker mortality respectively. With lower concentrations (1/2, 1/5 and 1/10 MFRC) the toxicity decreased, but the effect depended on the route of exposure. In addition to lethal effects, nests were also evaluated for sublethal effects after treatment with the seven MCAs at their respective MFRCs over 11 weeks. In these bioassays, only Botanigard and Serenade gave rise to a significant (P < 0.05) decrease in drone production. Sublethal effects on foraging behaviour were also evaluated, and only Botanigard at its MFRC delivered via treated sugar water induced negative effects. CONCLUSION: The results demonstrated that most of the MCAs tested can be considered safe for use in combination with B. terrestris, based on the International Organisation for Biological Control of Noxious Animals and Plants (IOBC) classification. However, some can be harmful, such as the biofungicide Serenade and the bioinsecticide Botanigard. Therefore, it is recommended that all should be tested before use in combination with pollinators. In this context, it is also advisable that these MCAs should be evaluated in more realistic field situations for the assessment of potentially deleterious effects on foraging behaviour.

Bumblebees can be used in combination with juvenile hormone analogues and ecdysone agonists.

This study examined the lethal and sublethal effects on the beneficial insect Bombus terrestris by two classes of insect growth regulators (IGRs) that are commercially used in agriculture to control pest insects. Three juvenile hormones analogues (JHAs) (pyriproxyfen, fenoxycarb and kinoprene) and two ecdysone agonists or moulting accelerating compounds (MACs) (tebufenozide and methoxyfenozide) were tested. The bumblebee workers were exposed to the insecticides via three different routes of exposure: dermally by topical contact, and orally via the drinking sugar water or the pollen. In the first series of experiments the IGRs were applied at their respective maximum field recommended concentration (MFRC). These risk hazard tests showed that the tested IGRs caused no acute toxicity on the workers, and any compound had an adverse effect on reproduction (production of males). In addition, larval development was followed in the treated nests compared with the controls. After application of the two MACs and the JHA fenoxycarb no adverse effects were observed on larval development. However, in the nests where the workers were exposed to the JHAs pyriproxyfen and kinoprene higher numbers of dead larvae were scored. These larvae were third and fourth instars, implying a lethal blockage of development before metamorphosis. In a second test, a series of dilutions was made for kinoprene, and these results revealed that only the MFRC caused a toxic effect on the larval development. On the other hand, kinoprene at lower concentrations (0.0650 mg ai/l) had a stimulatory effect on brood production. It was remarkable that ovaries of such treated dominant workers were longer and contained more eggs than in the controls. In a last experiment, the cuticular uptake was determined for a JHA and MAC to evaluate to what extent worker bees accumulate these classes of IGRs. Cuticular uptake ranged from 34 to 83% at 24 h after topical application. Overall, the obtained results indicate that the tested IGRs at their recommended concentration are safe to be used in combination with B. terrestris.