Synergistic negative effects between a fungicide and high temperatures on homing behaviours in honeybees.

Interactions between environmental stressors may contribute to ongoing pollinator declines, but have not been extensively studied. Here, we examined the interaction between the agricultural fungicide Pristine (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) and high temperatures on critical honeybee behaviours. We have previously shown that consumption of field-realistic levels of this fungicide shortens worker lifespan in the field and impairs associative learning performance in a laboratory-based assay. We hypothesized that Pristine would also impair homing and foraging behaviours in the field, and that an interaction with hot weather would exacerbate this effect. Both field-relevant Pristine exposure and higher air temperatures reduced the probability of successful return on their own. Together, the two factors synergistically reduced the probability of return and increased the time required for bees to return to the hive. Pristine did not affect the masses of pollen or volumes of nectar or water brought back to the hive by foragers, and it did not affect the ratio of forager types in a colony. However, Pristine-fed bees brought more concentrated nectar back to the hive. As both agrochemical usage and heat waves increase, additive and synergistic negative effects may pose major threats to pollinators and sustainable agriculture.

Using electric fields to control insects: current applications and future directions.

Chemical-based interventions are mostly used to control insects that are harmful to human health and agriculture or that simply cause a nuisance. An overreliance on these insecticides however raises concerns for the environment, human health, and the development of resistance, not only in the target species. As such, there is a critical need for the development of novel nonchemical technologies to control insects. Electrocution traps using UV light as an attractant are one classical nonchemical approach to insect control but lack the specificity necessary to target only pest insects and to avoid harmless or beneficial species. Here we review the fundamental physics behind electric fields (EFs) and place them in context with electromagnetic fields more broadly. We then focus on how novel uses of strong EFs, some of which are being piloted in the field and laboratory, have the potential to repel, capture, or kill (electrocute) insects without the negative side effects of other classical approaches. As EF-insect science remains in its infancy, we provide recommendations for future areas of research in EF-insect science.

'Inert' co-formulants of a fungicide mediate acute effects on honey bee learning performance.

Managed honey bees have experienced high rates of colony loss recently, with pesticide exposure as a major cause. While pesticides can be lethal at high doses, lower doses can produce sublethal effects, which may substantially weaken colonies. Impaired learning performance is a behavioral sublethal effect, and is often present in bees exposed to insecticides. However, the effects of other pesticides (such as fungicides) on honey bee learning are understudied, as are the effects of pesticide formulations versus active ingredients. Here, we investigated the effects of acute exposure to the fungicide formulation Pristine (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) on honey bee olfactory learning performance in the proboscis extension reflex (PER) assay. We also exposed a subset of bees to only the active ingredients to test which formulation component(s) were driving the learning effects. We found that the formulation produced negative effects on memory, but this effect was not present in bees fed only boscalid and pyraclostrobin. This suggests that the trade secret "other ingredients" in the formulation mediated the learning effects, either through exerting their own toxic effects or by increasing the toxicities of the active ingredients. These results show that pesticide co-formulants should not be assumed inert and should instead be included when assessing pesticide risks.

Decision-making: A new role for insect mushroom bodies.

Mushroom bodies are neural structures that are ubiquitous in insect brains, where they integrate sensory inputs to encode stimulus identity and meaning. A new study now adds action selection - decision-making - to those roles.

Comment on "Food wanting is mediated by transient activation of dopaminergic signaling in the honey bee brain".

Huang et al. (1) make an exciting claim about a human-like dopamine-regulated neuromodulatory mechanism underlying food-seeking behavior in honey bees. Their claim is based on experiments designed to measure brain biogenic amine levels and manipulate receptor activity. We have concerns that need to be addressed before broad acceptance of their results and the interpretation provided.

A mitotoxic fungicide alters post-ingestive glucose signals necessary for associative learning in honey bees.

The Proboscis Extension Reflex (PER) paradigm trains honey bees to associate an odor with a sugar reward and is commonly used to assess impacts on associative learning after exposure to pesticides. While the effects of some types of pesticides have been well-investigated, relatively little attention has been focused on fungicides that are applied to flowering crops. We have previously shown that consumption of field-relevant concentrations of the fungicide Pristine® (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) impairs honey bee performance in an associative learning assay, but the mechanism of its action has not been investigated. We hypothesized that Pristine® interferes with carbohydrate absorption and/or regulation, thereby disrupting the post-ingestive feedback mechanisms necessary for robust learning. To test this hypothesis, we measured hemolymph glucose and trehalose levels at five time points during the ten minutes after bees consumed a sucrose solution. Pristine®-exposed bees had elevated baseline glucose concentrations in the hemolymph relative to control bees. Hemolymph glucose levels rose significantly within five minutes of feeding in control bees, but not in Pristine®-fed bees. These data suggest that the post-ingestive feedback mechanisms necessary for robust learning are disrupted in bees that have consumed this fungicide, providing a plausible mechanistic explanation for its effects on learning performance in the PER assay. Pristine®-exposed bees may have elevated hemolymph glucose levels because the fungicide elicits an inflammatory response. These results provide additional mechanistic understanding of the negative physiological effects of mitotoxic fungicides on this important pollinator.

Assessing complex odor discrimination in mice using a novel instrumental patterning task.

Negative patterning tasks are a key tool to unveil the mechanisms by which stimulus representations are acquired-a central concern in Robert Rescorla's research. In these tasks, target stimuli are reinforced when presented individually (A+/B+) but not when presented in compound (AB-). The discrimination of single stimuli from their compound presentation is a challenge for theories of associative learning, because it cannot be explained by the simple accrual of associative strength. The present study examined the conditions under which mice learn this part-whole discrimination in olfactory stimuli using a novel instrumental methodology. In two experiments, reinforcement was contingent on distinct responses depending on whether a set of odor mixtures were presented in isolation or as a compound. Using C57BL/6 mice, Experiment 1 showed a mutual interference between learning a response to individual odors and learning a different response to those odors presented in compound. Using wild-type APP/PS1 mice (a control strain for a murine model of Alzheimer's disease), Experiment 2 replicated this interference and showed that it is stimulus-specific. These experiments show that the instrumental patterning task may not only complement Pavlovian negative patterning tasks but may also motivate its own questions on the representation of complex stimuli. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Seasonal variability in physiology and behavior affect the impact of fungicide exposure on honey bee (Apis mellifera) health.

Honey bee pollination services are of tremendous agricultural and economic importance. Despite this, honey bees and other pollinators face ongoing perils, including population declines due to a variety of environmental stressors. Fungicides may be particularly insidious stressors for pollinators due to their environmental ubiquity and widespread approval for application during crop bloom. The mechanisms by which fungicides affect honey bees are poorly understood and any seasonal variations in their impact are unknown. Here we assess the effects on honey bee colonies of four-week exposure (the approximate duration of the almond pollination season) of a fungicide, Pristine® (25.2% boscalid, 12.8% pyraclostrobin), that has been commonly used for almonds. We exposed colonies to Pristine® in pollen patties placed into the hive, in either summer or fall, and assessed colony brood and worker populations, colony pollen collection and consumption, and worker age of first foraging and longevity. During the summer, Pristine® exposure induced precocious foraging, and reduced worker longevity resulting in smaller colonies. During the fall, Pristine® exposure induced precocious foraging but otherwise had no significant measured effects. During the fall, adult and brood population levels, and pollen consumption and collection, were all much lower, likely due to preparations for winter. Fungicides and other pesticides may often have reduced effects on honey bees during seasons of suppressed colony growth due to bees consuming less pollen and pesticide.

Hyperbolic odorant mixtures as a basis for more efficient signaling between flowering plants and bees.

Animals use odors in many natural contexts, for example, for finding mates or food, or signaling danger. Most analyses of natural odors search for either the most meaningful components of a natural odor mixture, or they use linear metrics to analyze the mixture compositions. However, we have recently shown that the physical space for complex mixtures is 'hyperbolic', meaning that there are certain combinations of variables that have a disproportionately large impact on perception and that these variables have specific interpretations in terms of metabolic processes taking place inside the flower and fruit that produce the odors. Here we show that the statistics of odorants and odorant mixtures produced by inflorescences (Brassica rapa) are also better described with a hyperbolic rather than a linear metric, and that combinations of odorants in the hyperbolic space are better predictors of the nectar and pollen resources sought by bee pollinators than the standard Euclidian combinations. We also show that honey bee and bumble bee antennae can detect most components of the B. rapa odor space that we tested, and the strength of responses correlates with positions of odorants in the hyperbolic space. In sum, a hyperbolic representation can be used to guide investigation of how information is represented at different levels of processing in the CNS.

Alternative model systems for cognitive variation: eusocial-insect colonies.

Understanding the origins and maintenance of cognitive variation in animal populations is central to the study of the evolution of cognition. However, the brain is itself a complex, hierarchical network of heterogeneous components, from diverse cell types to diverse neuropils, each of which may be of limited use to study in isolation or prohibitively challenging to manipulate in situ. Consequently, highly tractable alternative model systems may be valuable tools. Eusocial-insect colonies display emergent cognitive-like properties from relatively simple social interactions between diverse subunits that can be observed and manipulated while operating collectively. Here, we review the individual-scale mechanisms that cause group-level variation in how colonies solve problems analogous to cognitive challenges faced by brains, like decision-making, attention, and search.

Unbalanced fatty acid diets impair discrimination ability of honey bee workers to damaged and healthy brood odors.

Nutrition supports social insect colonies by regulating both individual performance and colony growth. In honey bee colonies, task-related behaviors such as nursing and foraging are partially mediated by nutrition. Young workers (nurses) consume almost all of the pollen in the hive, while foragers consume mostly nectar. Pollen provides vital proteins and lipids, consumed by nurse bees for approximately 1 week post-eclosion. The role that lipids play in the physiology and behavior of adult bees is gaining significant attention. Recent research suggests that diets with balanced ratios of fatty acids increase olfactory learning in honey bees. Olfaction is crucial for young worker bees to perform brood care and cell cleaning behaviors, which is important for hive health and disease control. Thus, we targeted the early adult, pollen-feeding stage to examine how fatty acids affect cognition to hive-relevant odors. We fed young workers (days 0-9) diets balanced or unbalanced in their ratio of essential fatty acids (ω-6:3) sourced from pollen or cooking oils. We then measured their ability to learn healthy and damaged brood odors, as well as their ability to discriminate between the two. Workers fed balanced diets could learn and discriminate between brood odors better than workers fed unbalanced diets. Consumption of both diet types decreased with age, but their cognitive effects remained. These results suggest that diet affects young worker cognitive development, which may affect task-related behaviors and colony hygiene.

Novelty detection in early olfactory processing of the honey bee, Apis mellifera.

Animals are constantly bombarded with stimuli, which presents a fundamental problem of sorting among pervasive uninformative stimuli and novel, possibly meaningful stimuli. We evaluated novelty detection behaviorally in honey bees as they position their antennae differentially in an air stream carrying familiar or novel odors. We then characterized neuronal responses to familiar and novel odors in the first synaptic integration center in the brain-the antennal lobes. We found that the neurons that exhibited stronger initial responses to the odor that was to be familiarized are the same units that later distinguish familiar and novel odors, independently of chemical identities. These units, including both tentative projection neurons and local neurons, showed a decreased response to the familiar odor but an increased response to the novel odor. Our results suggest that the antennal lobe may represent familiarity or novelty to an odor stimulus in addition to its chemical identity code. Therefore, the mechanisms for novelty detection may be present in early sensory processing, either as a result of local synaptic interaction or via feedback from higher brain centers.

The active ingredients of a mitotoxic fungicide negatively affect pollen consumption and worker survival in laboratory-reared honey bees (Apis mellifera).

Recent observations of many sublethal effects of pesticides on pollinators have raised questions about whether standard short-term laboratory tests of pesticide effects on survival are sufficient for pollinator protection. The fungicide Pristine® and its active ingredients (25.2% boscalid, 12.8% pyraclostrobin) have been reported to have low acute toxicity to caged honey bee workers, but many sublethal effects at field-relevant doses have been reported and Pristine® was recently found to increase worker pollen consumption, reduce worker longevity and colony populations at field relevant concentrations (Fisher et al. 2021). To directly compare these whole-colony field results to more standard laboratory toxicology tests, the effects of Pristine®, at a range of field-relevant concentrations, were assessed on the survival and pollen consumption of honey bee workers 0-14 days of age. Also, to separate the effects of the inert and two active ingredients, bees were fed pollen containing boscalid, pyraclostrobin, or pyraclostrobin plus boscalid, at concentrations matching those in the Pristine® treatments. Pyraclostrobin significantly reduced pollen consumption across the duration of the experiment, and dose-dependently reduced pollen consumption on days 12-14. Pristine® and boscalid significantly reduced pollen feeding rate on days 12-14. Boscalid reduced survival in a dose-dependent manner. Consumption of Pristine® or pyraclostrobin plus boscalid did not affect survival, providing evidence against strong negative effects of the inert ingredients in Pristine® and against negative synergistic effects of boscalid and pyraclostrobin. The stronger toxic effects of Pristine® observed in field colonies compared to this laboratory test, and the opposite responses of pollen consumption in the laboratory and field to Pristine®, show that standard laboratory toxicology tests can fail to predict responses of pollinators to pesticides and to provide protection.

Heritable Cognitive Phenotypes Influence Appetitive Learning but not Extinction in Honey Bees.

Learning and attention allow animals to better navigate complex environments. While foraging, honey bees (Apis mellifera L.) learn several aspects of their foraging environment, such as color and odor of flowers, which likely begins to happen before they evaluate the quality of the food. If bees begin to evaluate quality before they taste food, and then learn the food is depleted, this may create a conflict in what the bee learns and remembers. Individual honey bees differ in their sensitivity to information, thus creating variation in how they learn or do not learn certain environmental stimuli. For example, foraging honey bees exhibit differences in latent inhibition (LI), a learning process through which regular encounter with a stimulus without a consequence such as food can later reduce conditioning to that stimulus. Here, we test whether bees from distinct selected LI genotypes learn differently if reinforced via just antennae or via both antennae + proboscis. We also evaluate whether learned information goes extinct at different rates in these distinct LI genetic lines. We find that high LI bees learned significantly better when they were reinforced both antenna + proboscis, while low LI and control bees learned similarly with the two reinforcement pathways. We also find no differences in the acquisition and extinction of learned information in high LI and low LI bees. Our work provides insight into how underlying cognition may influence how honey bees learn and value information, which may lead to differences in how individuals and colonies make foraging decisions.

A common fungicide, Pristine®, impairs olfactory associative learning performance in honey bees (Apis mellifera).

Although fungicides were previously considered to be safe for important agricultural pollinators such as honey bees, recent evidence has shown that they can cause a number of behavioral and physiological sublethal effects. Here, we focus on the fungicide Pristine® (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin), which is sprayed during the blooming period on a variety of crops and is known to affect honey bee mitochondria at field-relevant levels. To date, no study has tested the effects of a field-relevant concentration of a fungicide on associative learning ability in honey bees. We tested whether chronic, colony-level exposure at field-relevant and higher concentrations of Pristine® impairs performance on the proboscis extension reflex (PER) paradigm, an associative learning task. Learning performance was reduced at higher field-relevant concentrations of Pristine®. The reductions in learning performance could not be explained by effects on hunger or motivation, as sucrose responsiveness was not affected by Pristine® exposure. To determine whether Pristine®'s negative effects on learning performance were mediated at a specific life stage, we conducted a cross-fostering experiment that exposed bees to the fungicide either only as larvae, only as adults, or during both stages. We found that exposure across the entire life was necessary to significantly reduce learning performance, although non-significant reductions occurred when bees were exposed during just one stage. Our study provides strong evidence that Pristine® has significant sublethal effects on learning performance. As associative learning is a necessary ability for foraging, our results raise concerns that Pristine® could impair foraging abilities and substantially weaken colony health.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera).

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Colony field test reveals dramatically higher toxicity of a widely-used mito-toxic fungicide on honey bees (Apis mellifera).

Honey bees (Apis mellifera) and other pollinator populations are declining worldwide, and the reasons remain controversial. Based on laboratory testing, fungicides have traditionally been considered bee-safe. However, there have been no experimental tests of the effects of fungicides on colony health under field conditions, and limited correlational data suggests there may be negative impacts on bees at levels experienced in the field. We tested the effects of one of the most commonly used fungicides on colony health by feeding honey bee colonies pollen containing Pristine® (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) at four levels that bracketed concentrations we measured for pollen collected by bees in almond orchards. We also developed a method for calculating per-bee and per-larva dose. Pristine® consumption significantly and dose-dependently reduced worker lifespan and colony population size, with negative health effects observed even at the lowest doses. The lowest concentration we tested caused a 15% reduction in the worker population at an estimated dosage that was three orders of magnitude below the estimated LD15 values for previous acute laboratory studies. The enhanced toxicity under field conditions is at least partially due to activation of colonial nutritional responses missed by lab tests. Pristine® causes colonies to respond to perceived protein malnutrition by increasing colony pollen collection. Additionally, Pristine induces much earlier transitioning to foraging in individual workers, which could be the cause of shortened lifespans. These findings demonstrate that Pristine® can negatively impact honey bee individual and colony health at concentrations relevant to what they experience from pollination behavior under current agricultural conditions.

Early olfactory, but not gustatory processing, is affected by the selection of heritable cognitive phenotypes in honey bee.

Associative learning enables animals to predict rewards or punishments by their associations with predictive stimuli, while non-associative learning occurs without reinforcement. The latter includes latent inhibition (LI), whereby animals learn to ignore an inconsequential 'familiar' stimulus. Individual honey bees display heritable differences in expression of LI. We examined the behavioral and neuronal responses between honey bee genetic lines exhibiting high and low LI. We observed, as in previous studies, that high LI lines learned a familiar odor more slowly than low LI bees. By measuring gustatory responses to sucrose, we determined that perception of sucrose reward was similar between both lines, thereby not contributing to the LI phenotype. We then used extracellular electrophysiology to determine differences in neural responses of the antennal lobe (AL) to familiar and novel odors between the lines. Low LI bees responded significantly more strongly to both familiar and novel odors than the high LI bees, but the lines showed equivalent differences in response to the novel and familiar odors. This work suggests that some effects of genotype are present in early olfactory processing, and those effects could complement how LI is manifested at later stages of processing in brains of bees in the different lines.