by Anne L. Averill, University of Massachusetts/Amherst
CAP Updates: 22
Bumble bees are able to readily relocate their nest following a 1.0-2.0 km (0.6-1.2 miles) displacement, probably using landmarks. Results of our very preliminary homing bioassays, which we refined to evaluate impact of insecticides, indicate that bee size, floral resource structure, and complexity of surrounding landscape will influence results. I suggest that in intensive monocultures, bees may face a dual disadvantage when returning from foraging bouts since they must deal not only with featureless agricultural landscapes, but also with neurotoxic insecticides that may impair proper nervous system functioning, and thus, compromise orientation.
Studies have shown that native non-Apis bees can play an especially important role as pollinators in many crops and in fact, may provide more efficient pollination services in some crops, for example, squash, watermelon, cranberry, blueberry, and tomato. However, concern regarding the stability of pollination services has come to the forefront in the past decade. We are faced with a troubling situation where it is increasingly difficult to manage our key pollinator, Apis mellifera, and where an increasing number of studies report decline in the diversity of wild pollinator species (Potts et al. 2010). Recently, the status of bumble bees (Bombus) has become a focus of significant attention owing to reports of range reductions and local extinctions of several species (Cameron et al. 2011). Potential causes of decline in Bombus have been suggested, including land-use changes, pathogen introduction, and pesticides.
Insecticides and bees.
A major shift in both home and agricultural settings has been the startlingly rapid introduction of neonicotinoid and phenylpyrazole insecticides; these differ from most classic insecticides in that they are systemic in the plant and may be detected in pollen and nectar throughout the blooming period. As a consequence, bees could experience chronic exposure to them over long periods of time and at all stages of development (see the February 2011 CAP column by Marion Ellis for a review). Further, multiple exposures to multiple insecticides, perhaps along with high levels of fungicide applied over foraging bees during bloom, could result in synergistic impacts. To add insult to injury, honey bee studies have documented negative synergistic effects when imidacloprid (a neonicotinoid) and Nosema infection (a microsporidian pathogen) are combined (Alaux et al. 2010, J. Pettis, unpublished).
Until recently, when a pesticide moved through registration, most studies and guidelines focused on the dose that resulted in death of the honey bee, with much less attention given to sublethal effects, such as abnormal foraging behavior resulting from nerve poisons. An assumption was often made that toxicity would be parallel for all bee species. In reality, this has not proven to be true in many comparative studies, and we should expect a range of impacts of a pesticide across bee species since life history traits and size vary so widely (Brittain and Potts 2011). For example, in contrast to honey bees, bumble bee colonies are comprised of only 10-100s of workers and are annual, being founded by a queen in the spring. While honey bee larvae primarily are fed secretions from nurse bees, bumble bee larvae develop directly on unprocessed pollen. It is possible that this raw bumble bee diet may end up having a higher concentration of pesticide contamination than honey bee larval diet (Fisher and Moriarty 2010). (On the other hand, metabolites could show up in honey bee worker secretions, and these are often more toxic than the parent chemicals.) Additionally, bumble bees vary in size across species and importantly, exhibit more size variation within a colony than any other bee species; workers can vary in mass by 8-10 fold in a given colony (Goulson et al. 2002, Jandt and Dornhaus 2009). Smaller bees are often more susceptible to insecticides owing to their higher surface area to volume ratio. As a result, an LD50 value (the dose causing mortality of 50% of the test subjects), which is expressed as dose/bee, would require accompanying bee size information in order to make the most useful comparisons.
Figure 1. Bumble bees form annual colonies and can be important pollinators for several crops such as blueberry, tomato, and cranberry.
Many studies of bees have shown sublethal effects of neonicotinoids, such as imidacloprid, or other neurotoxic insecticides, including impaired learning, delays in foraging, and reduction in orientation abilities, but it has been difficult to determine if the doses utilized in tests are biologically relevant. However, studies working at lower and lower doses are showing deleterious outcomes in more sophisticated and natural bioassays. In greenhouse studies of bumble bees, Mommaerts et al. (2010) found that behavioral bioassays that included foraging were 3-10 times more sensitive when assessing sublethal effects of imidacloprid. We went a step further and investigated orientation of bumble bees in field settings. Here, I will describe our efforts to identify factors that could influence consistency of such field tests and then I report our preliminary data on the impact of a sublethal dose of insecticide on the ability of bumble bees to relocate their nest.
How do foraging bees relocate their nest?
When a bee leaves the nest or a newly discovered food resource for the first time, they back away from the site in a series of increasing arcs. It is thought that this allows them to record the scene around the nest with defined vantage points and to acquire information about distances between objects (Zeil et al. 1996). Based on their Bombus studies where bees were released at points up to 15 km from the nest, Goulson and Stout (2001) believe that the most likely homing mechanism used by the bees when they are artificially displaced (i.e. carried away from the nest and released at a distant point) would be a systematic search around the release site; they would fly out further and further from the release point until they recognized familiar landmarks, which would then be used to locate their nest. If this holds true, then the homing success of a displaced bee will depend on its experience and familiarity with the areas around the nest. Individuals that varied the distances and locations where they collect pollen and nectar should be more likely to find the nest than those that repeatedly visit the closest patches.
Can we identify important factors that influence the consistency of our homing tests?
General methods: Bombus impatiensCresson (Common Eastern bumble bee) colonies with 100-200 workers were purchased from a mass-rearing program (Koppert Biological Systems, Howell, MI) and were simultaneously established inside a lab building, one located in the SE Massachusetts growing region and the other on the UMass Amherst campus. Bees were allowed to exit the nest and return from foraging sites via clear vinyl tubes attached to the nest box (Figure 2). Colonies were given two weeks to adjust prior to tests.
Figure 2. Commercially-available bumble bee nests, contained in two layers of packaging, were fitted with clear plastic tubing so that workers could forage and return.
Landscape effects on homing. We have completed only a single replicate for Test 1 and Test 2 (so data are preliminary, no statistical analysis has been applied, and thus, our conclusions are speculative). Test 1. Effect of nearby mass-flowering crops. In southeastern Massachusetts, during mass flowering of cranberry, our surveys of other flowering plants showed that the extensive carpets of cranberry flowers (Figure 3) are more attractive than the sparsely distributed wild plants or managed plantings in these agroecosystems. Without a doubt, we expected that during bloom, the East Wareham experimental bees (see description below), whose nests were located fewer than 50 meters from a large commercial bog, would forage nearby and show poor homing ability following a distant displacement. Following bloom, we believed that bees would have more experience searching in near and far habitats for patchy and rare floral resources, and thus would have greater homing success following displacement.
To test this, boxes were established at the onset of cranberry bloom in a lab building at the UMass Cranberry Station in East Wareham, MA (designated as an “Agricultural” site) and new nests were established again in August, when bloom was completely over. As bees exited nests early in the morning, individuals were collected from four hives and randomly divided into one of three release distances: 0.5, 1.0 and 2.0 km (in a NE transect away from the nests). Bees were cooled in the freezer, marked with an identifying color dot for the release distance, allowed to recover, carried to one of the three given distances, and released. Returning bees were captured as they arrived to enter the nest; individuals were weighed and examined for a pollen load. During cranberry bloom, only 33% and 8% of bees that were released at 1 and 2 km, respectively, returned to the nest, while more than 60% of those released 0.5 km (0.3 miles) returned (Figure 4). In contrast, when new nests were established later in the summer (following the completion of cranberry bloom) the majority of bees returned from both the 0.5 and 1 km release sites, and close to a third of foragers returned successfully from the 2 km release. Presumably, these bees foraged more widely and distantly in the habitat and were able to recognize and utilize more landmarks during the homing process.
Test 2. Effect of landscape complexity: We compared homing success for bees displaced in landscapes that differed hugely in complexity: the Cranberry Station (‘Agricultural’ site) and the University of Massachusetts campus site in Amherst, MA (“Urban” site) (Figure 5). All methods for release at the two locations were as identical as possible and were carried out post-bloom. Cohorts of marked bees were displaced at 0.5, 1.0, and 2.0 km from the nest and recaptured as they returned. For bees in the agricultural landscape, homing success dropped off sharply with distance; in contrast, there was no difference in homing success as a function of displacement distance in the more complex urban landscape (Figure 6), which is dense with landmarks. For all distances combined, a clear difference in the speed of return was observed: at the urban site, 37.7% (23/61) of bees returned within the first hour following release and at the agricultural site, only 15.0% (9/60) of bees returned within this interval. These data suggest that homing may become increasingly difficult in homogenous landscapes such as would be found in monocultures and agriculturally-intensive areas. Ideally, we would have liked to compare the agriculturally-intense setting with diversified farm systems within natural habitats , but this was not possible.
Effect of sublethal dose of imidacloprid on homing in bumble bees.
While foraging, bees can be at risk due to pesticide treatments via several routes, for example by (1) ingestion of contaminated nectar, pollen, or guttation fluid (exudation of xylem fluid on leaves), (2) by direct contact during the pesticide treatment, or (3) by exposure to treated surfaces. All of these contacts could occur as the result of a single exposure event (acute exposure) or by repeated exposure events (chronic exposure).
Insecticide treatment: We started with the simplest exposure route, direct contact, by making a single topical application of a sublethal dose of imidacloprid to bumble bees set up in the lab as described above. We reasoned that this approach could give insight into the potential outcome of a sublethal dose when a forager was oversprayed by an insecticide application or that arrived on wet foliage following a spray. Technical grade imidacloprid was used. Insecticide stock was prepared via serial dilutions of compound in acetone and 5 ng in 5ml were applied to treated bees (or 5ml solvent alone = control bees). [To imagine the units we were working with: ng = nanogram and is one billionth of a gram (a gram could be visualized as the weight of ¼ teaspoon of sugar)]. Marletto et al. (2003) reported that the LD50 for medium-sized Bombus terrestris was 20 ng/bee. For our Bombus impatiens, in earlier studies we established an acute contact LD50 of 13 ng/bee; thus, our initial sublethal dose of 5 ng was high, or 40% of the LD50. Effect of body size: Because the foraging bees exiting the nests varied greatly in size (and thus, would likely show variation in susceptibility), using body mass, we created two test groups, large (.22-.29 g) and small (0.12-0.19 g).
Workers exiting the nest in early morning were captured, cooled, measured, marked, and either treated with insecticide in acetone or treated with acetone only. All bees were transported to a release site located 0.5 km away and allowed to fly away. The test was replicated three times on three different days for a total of 24 bees per treatment. Fewer treated bees returned to the nest successfully, demonstrating that the sublethal dose of 5 ng imidacloprid impaired the ability of foragers to orient to landmarks when artificially displaced. Further, this effect was more pronounced for smaller bees (significant effect of treatment and a significant interaction between size and treatment: p < .01, factorial ANOVA). Our next step in our homing studies will be to move to lower treatment rates, to assess additional new insecticide chemistries that function as neurotoxins, and to begin ingestion studies based on incoming data that establish field levels of systemic insecticides in pollen and nectar.
Figure 3. Mass flowering of cranberry. Photo: Cranberry Coast Chamber of Commerce
Limitations of our studies. Several issues can be raised. First, we are working with lab-selected and reared colonies so caution must be applied when extrapolating our results to wild populations. Second, for the insecticide treatments, optimally (but this is not possible) we would capture marked, foraging bumble bees at sites distant from the nest and treat them, rather than capturing them as they left the nest. Under the optimal scenario, the bee would have experienced the landscape on the outward trip from nest to foraging site and have the advantage of a learned route with landmarks. Directionality of release locations could be important (Pahl et al. 2011) and was not considered. Finally, determinations of the actual incidence of insecticide contact in the field, particularly for ingestion of contaminated nectar and pollen (which we have not yet addressed) will be useful to make demands that more thorough evaluation of risk and modification of use patterns of neurotoxic insecticides should be mandated.
Acknowledgements: This work was ably and patiently carried out by Zach Scott, Andrea Couto, Sunil Tewari, Martha Sylvia, and Ken LeFebvre, all from the University of Massachusetts/Amherst .
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