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What other cognitive behaviors in bees, outside of navigation, are affect by neonicotinoids

What other cognitive behaviors in bees, outside of navigation, are affect by neonicotinoids



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It is conclusive that neonicotinoids alter navigation in bees when locating food sources. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091364

But if so, shouldn't cognition also be affected? And if it is affected, then wouldn't the effect on the hive be even more pronounced due to inaccurate bee dances and an overall delay/inability in finding food source? Shouldn't this be conclusive in explaining neonicotinoids to be a contributor to colony collapse disorder?


It is assumed that this effect of locomotive behavior IS an effect on cognition (1-4). Navigation requires snapshot memory, learning, and attention (2-4). You asked if this effect of neonicotinoids on cognition manifests itself through other vital behaviors. The short answer, yes. Specifically neonicotinoids are shown to effect mating, queen behavior, and foraging short and long distance (5-7). It is suggestive that neonicotinoids are a contributing factor in collapsing colonies with both meta analysis and direct evidence of effects on behavior (5-8). I think you are more questioning why people have not done anything about this issue or more definitive in their action against colony collapse. That is more of a political issue. Since the pesticide does not have as many human health effects at levels that cause bee collapse, the policies to resolve this issue are not as clear. EPA has discussed making restrictions regarding use the use of neonicotinoids, but they will still be used. However, they have actually been banned in France due to these effects. Hope that answered your question!

Sources

  1. Honey Bee Behaviors doi:10.1016/S1364-6613(00)01601-6
  2. Bee Navigation and Memory doi:10.1006/anbe.1997.0574
  3. Learning and Memory in Bees doi: 1146/annurev.ne.19.030196.002115
  4. Behavioral Neuroscience ISBN: 978-0-87893-092-0
  5. Bees, neonicotinoids, and Dance Behavior Effects doi: 10.1242/jeb.068718
  6. Bees, neonicotinoids, and Foraging Behavior Effects DOI: 10.1046/j.1365-2435.2000.00443.x
  7. Queen and Colony Effects DOI: 10.1126/science.1215025
  8. Colony Collapse meta study DOI: 10.1007/s10646-010-0566-0

Study shows pesticide exposure can dramatically impact bees' social behaviors

A bumblebee (Bombus impatiens) worker foraging outdoors, outfitted with a unique tracking tag (BEEtag). Credit: James Crall

For bees, being social is everything.

Whether it's foraging for food, caring for the young, using their bodies to generate heat or to fan the nest, or building and repairing nests, a bee colony does just about everything as a single unit.

While recent studies have suggested exposure to pesticides could have impacts on foraging behavior, a new study, led by James Crall, has shown that those effects may be just the tip of the iceberg.

A post-doctoral fellow working in the lab of Benjamin de Bivort, the Thomas D. Cabot Associate Professor of Organismic and Evolutionary Biology, Crall is the lead author of a study that shows exposure to neonicotinoid pesticides—the most commonly-used class of pesticides in agriculture—has profound effects on a host of social behaviors.

Using an innovative robotic platform to observe bees' behavior, Crall and co-authors including de Bivort and Naomi Pierce, the Sidney A. and John H. Hessel Professor of Biology, showed that, following exposure to the pesticide, bees spent less time nursing larvae and were less social that other bees. Additional tests showed that exposure impaired bees ability to warm the nest, and to build insulating wax caps around the colony. The study is described in a November 9 paper in Science.

In addition to Crall, de Bivort and Pierce, the study was co-authored by Callin Switzer, Ph.D. '18, Stacey Combes from UC Davis, former Organismic and Evolutionary Biology research assistants Robert L. Oppenheimer and Mackay Eyster and Harvard undergraduate Andrea Brown, '19.

"These pesticides first came into use around the mid-1990s, and are now the most commonly-used class of insecticide around the globe," Crall said. "Typically, they work through seed treatment—high concentrations are dosed on seeds, and one reasons farmers and pesticide companies like these compounds is because they are taken up systemically by the plants. so the idea is they provide whole-plant resistance. But the problem is they also show up in the pollen and nectar bees are feeding on."

Over the past decade, Crall said, a number of studies have linked pesticide exposure with disruptions in foraging, "but there were reasons to suspect that wasn't the whole picture."

"Foraging is only a part of what bumblebees do," Crall said. "Those studies were picking up on the important effects these compounds were having on what's going on outside the nest, but there's a whole world of really important behaviors going on inside. and that's a black box we wanted to open up a bit."

Automated tracking of nest workers in a bumblebee (Bombus impatiens) colony. Credit: James Crall

To do it, Crall and colleagues developed a unique, benchtop system that allowed them to track the activity of bees in as many as a dozen colonies at a time.

"What we do is put a black and white tag with a simplified QR code, on the back of each bee," he said. "And there's a camera that can move over the colonies and track the behavior of each bee automatically using computer vision. so that allows us to look inside the nest."

Using the system, Crall and colleagues were able to dose specific, individual bees with the pesticide and observe the changes in their behavior—less interaction with nest-mates and spending more time on the periphery of the colony—but those experiments are limited in several important ways.

"One is physiological," Crall said. "Even though we were giving the bees realistic doses of pesticide, drinking your daily allotment of coffee in five minutes is going to be different than spreading it out over the course of the day, so giving one big dose might not be totally realistic. The other important one is that a bee colony is a functional unit. It doesn't make sense to treat individuals, because what you're losing when you do that is the natural social structure of the colony."

With the robotic system, however, researchers can treat an entire colony as a single unit.

Each of the system's 12 units, Crall said, houses a single colony where bees have access to two chambers—one to mimic the nest and the other to act as a foraging space.

"That lets us do multiple, colony-level exposure, and to do continuous monitoring," Crall said. "We think this is much closer to how their natural behavior works, and it also allows us to automate behavioral tracking across multiple colonies at the same time."

Just as in earlier studies, Crall said, exposed bees showed changes in activity levels and socialization, and spent more time on the fringes of the nest, but the tests also showed that the results were strongest overnight.

"Bees actually have a very strong circadian rhythm," Crall explained. "So what we found was that, during the day, there was no statistically-observable effect, but at night, we could see that they were crashing. We don't know yet whether (the pesticides) are disrupting circadian gene regulation or if this is just some, maybe physiological feedback. but it suggests that, just from a practical perspective, if we want to understand or study these compounds, looking at effects overnight matters a lot."

Manual feeding of a bumblebee (Bombus impatiens) worker during acute exposure trials. Credit: James Crall

Additional experiments, in which temperature probes were placed inside outdoor hives, suggested pesticides have profound effects on bees' ability to regulate temperatures inside the nest.

"When temperatures drop, bees lock their wings down and shiver their muscles to generate heat," Crall said. "But what we found was that, in control colonies, even as the temperature fluctuated widely, they were able to keep the temperature in the colony steady to within a few degrees. But the exposed bees, they pretty dramatically lose the capacity to regulate temperature."

In addition to disrupting bees' ability to directly heat or cool the nest, the experiment also revealed that pesticide exposure impacted bees' ability to build an insulating wax cap over the colony.

"Almost all of our control colonies built that cap," Crall said. "And it seems to be totally wiped out in the pesticide-exposed colonies, so they lose this capacity to do this functional restructuring of the nest."

Going forward, Crall said, there are some additional questions raised by the study that he hopes to address.

"This work—especially on thermoregulation—opens up a new set of questions, not just about what the direct effects of pesticides are, but how those pesticides impair the ability of colonies to cope with other stressors," he said. "This work suggests that, in particularly extreme environments, we might expect the effects of pesticides to be worse, so it changes both how we go about practically testing agro-chemicals in general, but it points to specific questions about whether we might see stronger declines in certain environments."

Taken together, Crall believes the findings point to the need for tighter regulation of neonicotinoids and other pesticides that may be impacting bees.

"I think we're at a point where we should be very, very concerned about how the ways in which we're changing the environment is undercutting and decimating insect populations that are important not only for the function of every ecosystem. but that are very important for food production," he said. "Our food system is becoming more and more pollinator-dependent over time—today about a third of food crops are dependent on pollinators, and that's only rising. Up until now, we've had this abundant, natural gift of pollinators doing all this work for us, and now we're starting to realize that isn't a given, so I think we should be very worried about that."


Neonicotinoid pesticide affects foraging and social interaction in bumblebees

In a plastic, lasercut box blacked out with paint and lit with red light, worker bumblebees (Bombus impatiens) go about their daily activities: interacting with fellow adults, extracting food from honey pots, feeding larvae, and occasionally venturing out to forage for nectar. While this nest is far from normal, the bees that live here have adapted to their new space remarkably well. Still, all is not well within the nest, and not because of its strange form. Some bees have abandoned their daily patterns and are spending more time alone, on the periphery. Others are spending less time caring for the utterly dependent larvae that will become the next generation of bumblebees.

Within the nest, the chaotic center of bumblebee life, social behavior and interactions are crucial for bee population health and the production of young. When social behavior and the care of young changes, population numbers become more susceptible to decline. James Crall, a postdoc with the Planetary Health Alliance at Harvard University, graduate student Callin Switzer and colleagues have linked these changes in social behavior with sublethal exposure to the neonicotinoid pesticide, imidacloprid.

For their study, Crall developed an 'automated behavioral tracking system' that allows a computer connected to cameras within the nest to recognize individual bees and create data points that indicate position and proximity to others. "Bumblebee nests are not the organized, beautiful geometry of the honeybee," said Crall. Instead, "they're more a hodge-podge of food and larvae in a pile in the middle of the nest space." This automated tracking system allowed Crall to see into "messy, complex, realistic, individual scenes" and could be adapted for use in natural environments.

While it might seem like the hardest part of this experiment would be development of a tracking system, Crall said the process of tagging each bee was both an art and a science, a "race against time" to glue on tags before the bees woke up, and "by far the hardest and slowest part of the experiment." Tagging a colony of bees could consume entire days, while bee movement within nests was only recorded for a few hours. After tagging, bees were observed before and after exposure to imidacloprid. Crall then evaluated millions of data points to assess behavioral changes among treated bees. He found that bees exposed to the pesticide reduced the frequency of brood care and tended to gravitate towards the perimeter of the nest, becoming less social.

Outside the nest, this neonicotinoid also has significant effects on pollination and foraging behavior. Callin Switzer, a PhD student at Harvard University, worked to study the effects of imidacloprid on pollination behavior. Specifically, Switzer focused on buzz pollination, the ability of bumblebees to forage on and pollinate certain types of plants, using vibrations. Before exposing bees to imidacloprid, Switzer recorded the sound of bees foraging on tomato blossoms. These same bees were then exposed to the neonicotinoid and allowed to resume foraging. However, bees exposed to imidacloprid, at doses similar to those encountered in a single day, were less likely to resume foraging than unexposed bees.

Imagine it's summer, and in a field by the side of the road, rows on rows of tomato plants wait to be pollinated and produce their delicious fruit. These plants reproduce more following buzz pollination, a service eastern bumble bees are uniquely equipped to provide. However, these tomato plants are covered in imidacloprid, and when bumblebees forage here, they are exposed to sublethal levels of this pesticide. As the season progresses and exposure to imidacloprid increases, bees are still present, but they begin to forage less, don't care for their young as often, and social interactions change. Outside the nest, a decrease in foraging by affected bumblebees could contribute to lessened crop production and colony food supplies. Within the nest, altered social networks and a decrease in caring for young could lead to population declines in future generations. As the single most important native pollinator species in North America, continued use of the neonicotinoid imidacloprid could have far-reaching effects on the survival of the Common Eastern Bumblebee and the plants they pollinate.

Callin Switzer and James Crall are presenting their research separately at the Society for Integrative and Comparative Biology's 2017 Annual Meeting in New Orleans, Louisiana. Switzer's research was recently published in Ecotoxicology as "The neonicotinoid pesticide, imidacloprid, affects Bombus impatiens (bumblebee) sonication behavior when consumed at doses below the LD50."


2 MATERIALS AND METHODS

2.1 Scope and search strategy

We focused upon olfactory learning and memory, which are typically assessed in bees through an olfactory proboscis extension reflex paradigm (hereafter PER). During a PER experiment, a harnessed bee learns to associate a previously unrewarded scent with sucrose. Bees initially exhibit proboscis extension as an unconditioned response (UR) to antennal contact with sucrose (the unconditioned stimulus US). When this contact is paired with a scent (the conditioned stimulus CS), the bee learns to extend its proboscis in response to the scent alone (a conditioned response CR). Typically, PER-based experiments that relate to pesticides use an absolute conditioning paradigm (where bees learn to associate only one scent with sucrose) rather than differential conditioning (where one scent is rewarded and an alternative is not Stanley, Smith, & Raine, 2015 ). Although other paradigms to test learning and memory (e.g., free-flying association, spatial learning, aversive learning, or tactile learning [Bernadou, Démares, Couret-Fauvel, Sandoz, & Gauthier, 2009 Tan et al., 2014 Samuelson, Chen-Wishart, Gill, & Leadbeater, 2016 Zhang & Nieh, 2015 ]) are available and widely used in the cognitive literature, only a very small number of studies have used such methods to assay how pesticides influence performance (see Section 4 Bernadou et al., 2009 Samuelson et al., 2016 Zhang & Nieh, 2015 ). In contrast, the PER paradigm is the most commonly used methodology to assess bee learning and memory and thus provides an obvious target for our study.

We used Web of Science and Google Scholar as search databases (search performed in April 2018). The search criteria used in Web of Science were (“pesticide*” OR “insecticide*” OR “neonicotinoid*”) AND (“bumblebee*” OR “bumble bee*” OR “honey bee*” OR “honeybee*” OR “bee*” OR “apis” OR “bombus”) AND (“learning” OR “memory” OR “PER” OR “cognition” OR “proboscis extension reflex” OR “proboscis extension response”). After the Web of Science search we used the same key words in Google Scholar and checked the first 200 results, which yielded three additional papers (Figure S1). Twenty-three papers remained eligible after title and abstract screening, and applying inclusion criteria (see below and Table S1). All 23 papers had their reference lists examined and we did not find any additional data.

2.2 Inclusion criteria, data extraction, and final database

To be included in the meta-analysis, a study had to involve oral exposure of bees to a pesticide followed by an assay of learning and/or memory via a PER conditioning paradigm. Studies were excluded if they did not contain a control group (no pesticide exposure) or if we were unable to extract the means, the standard deviations, and the sample sizes for both the control and the treatment groups. Some raw data were available online (N = 3), but in most cases (N = 17) the means and standard deviations could be extracted from graphs using WebPlotDigitizer (https://automeris.io/WebPlotDigitizer/). In cases where information was not available, some authors were successfully contacted (N = 3). We excluded experimental groups where the bees had been exposed to multiple stressors (e.g., both parasites and pesticides), as we could not be sure which stressor was potentially causing an effect. In all studies included in the analysis, bees were tested either directly or 24 h after pesticide exposure. We excluded one study where the postexposure testing period varied (with delays of up to 11 months Table S1). After sensitivity analysis (see below) the 23 papers included in the final database (see Table S1) yielded 104 effect sizes for the influence of pesticides on learning ability from 23 papers and 167 effect sizes from 19 papers for the influence of pesticides on memory. These studies were published between 2009 and 2017.

PER experiments use varying criteria to assess learning performance, including the number of trials in which the bee responded to the CS, the first trial in which it responded, or mean performance in a specified batch of trials. For example, Stanley, Smith, et al. ( 2015 ) used 15 learning trials (trials in which the UR and the CS are paired) per condition, whereas Piiroinen, Botías, Nicholls, and Goulson ( 2016 ) tested their bees over 10 trials. To enable direct comparison, we redefined learning across studies as the proportion of bees that responded positively to the CS by the final learning trial (intertrial interval mean = 8.17 ± 5.6). Similarly, we collated memory data (the number of bees responding to the CS) from all reported time lengths (range: 10 min–48 h) into two categories that approximate short- and long-term memory (see below). Note that these timings reflect neurologically distinct processes in bees, the transition from short- to long-term memory being translation-dependent (reviewed in Menzel, 2012 ).

2.3 Potential moderators

Moderators are used in meta-analysis to investigate the sources of variation in effect sizes between studies (Koricheva et al., 2013 ). Our meta-analysis included the following as potential moderators of the size of the effect that pesticide exposure had on learning and memory: pesticide exposure regime (chronic or acute), dosage (field realistic or above), pesticide type (neonicotinoid or other), and genus (Apis or Bombus). For the memory data, we also included short (<24 h) and long-term (≥24 h) memory retention as a potential moderator (see below for full models). The treatment was considered acute when the bees were exposed to one dosage of pesticide and chronic when the bees were repeatedly exposed over a sustained period of time, which varied between experiments from 4 days (Williamson & Wright, 2013 Yang, Chang, Wu, & Chen, 2012 ) to 24 days (Stanley, Smith, et al., 2015 ).

The definition of a field-realistic dose is highly contentious and the toxicity of different pesticides varies. To standardise this, we categorized dosages as field-realistic or above based on pesticide concentrations in nectar, pollen, honey, and bee-bread extracted from the following sources: Bonmatin et al. ( 2015 ), Glaberman and White ( 2014 ), Sanchez-Bayo and Goka ( 2014 ). Where more than one estimate was available for a given pesticide we took the mean value (see Table S2 for individual pesticides). For the acute dosages, the nectar pesticide concentration data were further combined with the mean amount of nectar that bees are able to ingest in one foraging bout (40 ng for honeybees 37.7 ng for bumblebees Table S3) to calculate the field realistic dose (Cresswell, 2011 Samuelson et al., 2016 ). Dosages higher than the above thresholds were considered not field realistic.

2.4 Meta-analysis

All analyses were conducted in R (version 1.0.136) using the package metafor (Viechtbauer, 2010 ). Data for learning and memory were analysed separately. We used standardized mean difference in bee learning ability or memory between the control groups and the treatment groups (Hedges’ d) as a measure of effect size (calculated using “escalc” function in metafor). For both datasets, we fitted random effects models to calculate the grand mean effect as well as the group means (e.g., effects of acute vs. chronic exposure). The restricted maximum likelihood approach (REML) was used to estimate the parameters of the meta-analysis models. For each of the two datasets, meta-regression was then used to explore the sources of variation in effect sizes by including all the moderators (see above) within a single model. Pesticide type was not included in these models because a subset of studies simultaneously exposed bees to more than one pesticide (Williamson, Baker, & Wright, 2013 Williamson & Wright, 2013 ), which would have led to these studies being dropped from the analyses (for full list of pesticides in meta-analysis see Table S2). Consequently, we analysed pesticide-type in a submodel that excluded these studies. “Study” was included as a random factor in all the models to control for potential nonindependence of multiple effect sizes from the same study.

We initially included in the analysis results from studies where bees were exposed to pesticides as larvae. However, there were very few of these (three studies for learning data and two studies for the memory data) and we found that the overall effect of pesticides on bee learning when these studies were included in the overall analysis was much stronger (d = −0.60, 95% CI = −0.90 to −0.30), whereas the overall effect of pesticides on bee memory was similar (d = −0.24, 95% CI = −0.28 to −0.20) compared to the effects based on the analysis when larval data were excluded from the analysis (see Section 3 for comparison). Thus, to preclude bias, we removed these studies from subsequent analyses. Furthermore, given the small number of studies conducted on bumblebees compared to honeybees, we conducted sensitivity analysis with studies that used honeybees only (see Figure S2). Within this analysis we also compared the impact of pesticides between the European (Apis mellifera) and the Asian honeybee (Apis cerana) (see Figure S2). We also re-ran the overall analysis without studies that used multiple pesticides (learning n = 2 and memory n = 2) and the results did not change (see supplementary material). We tested whether the number of learning trials undergone by the bees influenced the results and found no significant effect (p = 0.15) and thus we did not include this factor in the overall model. To test for any potential publication bias, a trim-and -fill technique was used on both the learning and memory data (Duval & Tweedie, 2000 ).


Honey bees lose sleep after ingesting pesticides, leading to greater stress and lower hive survival rates

Neonicotinoid ingestion alters circadian locomotor rhythms of honey bee foragers in light/dark and full darkness. Representative actograms of forager bees showing (A) control rhythmic activity, (B) loss of rhythms following ingestion of clothianidin (140 ppb) and altered locomotor rhythm patterns after ingestion of (C) thiamethoxam (140 ppb) and (D) clothianidin (140 ppb). Credit: Vanderbilt University

Bees that ingest nonlethal levels of popular pesticides resembling nicotine, known as neonicotinoids, are losing sleep, according to new research from Vanderbilt University. That disruption of their circadian rhythm causes honey bees to lose their sense of time and navigation, leading to broader stress within highly social bee populations and lower hive survival rates.

There has long been a mysterious connection between neonicotinoid pesticides and their lethal effect on bees. Just as the public began to notice a decline in bee populations, these pesticides took off as a plant maintenance technique. While the relationship seems logical, it had not been proven. Research led by Doug McMahon, Stevenson Chair of Biological Sciences, sought to explore the connection.

The article, "Neonicotinoids disrupt circadian rhythms and sleep in honey bees" was published in the journal Scientific Reports on Oct. 21.

"I was thinking about honey bee disappearances and it clicked—if pesticides are killing bees indirectly but we don't know exactly how, maybe it's because they're getting physically lost," said Michael Tackenberg, the postdoctoral scholar in the McMahon lab whose interest prompted the project.

Joined by collaborators—including Manuel Giannoni-Guzmán, another postdoctoral scholar in the McMahon lab—the team maintained healthy beehives to conduct experiments that explored how a widely used agricultural pesticide sold in home improvement stores prevents bees from getting the rest they need to thrive. A single bee can pollinate up to 5,000 flowers a day. Their combined efforts support a third of the world's food crop production.

In a series of experiments that exposed the bees to constant light, constant darkness and light and dark cycles, the researchers found a surprising mechanism by which the pesticide acts. Constant light conditions disrupted the circadian rhythm in 28 percent of bees. When levels of pesticides common in flower nectar and pollen were added to the bee's food supply, the number jumped to up to 46 percent. "Graphically, normal circadian rhythms look like steady waves," said Giannoni-Guzmán, the paper's co-first author. "When we observed bees that consumed neonicotinoids over several days, we saw a loss of waves, movement at random times or signs of barely any sleep at all."

Exploring this amplified disruption through mass spectroscopy, a technique that identifies and measures chemical compounds within molecules, the team found that neonicotinoids accumulate in the bee brain, disrupting circadian clock neurons.

"We have seen how neonicotinoids disrupt honey bees' biological clocks so that many no longer have regular sleep-wake rhythms," McMahon said. "The bees that do have irregular sleep-wake rhythms are sleep deprived and skewed in their alignment in time and environment."

Like people who don't get enough sleep, bees can't function as well if they are tired and disoriented. "Beyond sleep disruption, we know that honey bees rely on their internal sense of time and the position of the sun," said Tackenberg, also the paper's co-first author. "If they have an incorrect sense of time their ability to effectively navigate is hindered. It stands to reason that if a bee's internal sense of time is disrupted or altered it could affect learning, memory and foraging efficiency—even outside of reduced capacity from sleep disruptions." This work contributes to the body of evidence shaping how U.S. policymakers regulate the multi-billion-dollar farming and agriculture industries, which rely heavily the natural ability of bees to pollinate crops.

The team intends to look further into the mechanistic level of their findings by investigating the neural circuits of honey bees and the influence of neonicotinoids at the molecular level. "Since we now understand that the disruptive effect of pesticides is on a bee's circadian rhythm, there may be a way to help these important creatures reinforce and maintain their clock function in the face of this challenge," McMahon said.


The pollination services provided by bees are vitally important for ecosystem functioning and crop production. However, in recent decades, numerous reports have shown extensive losses of honeybee colonies and a decline in numbers of wild bees, with negative consequences for terrestrial ecosystems, the economy, and food security.

These losses have been attributed to many stress factors, including pesticide exposure, habitat loss or degradation, invasive species, predators, parasites, diseases, and climate change. These factors do not act alone and often show synergistic interactions that are difficult to predict.

In this Special Issue, we would like to publish original, theoretical or empirical research, reviews, quantitative meta-analyses or perspective articles focusing on how stress factors affect the health of managed and wild bees and on the defense mechanisms adopted at the individual level and, in the case of social species, also at the colony level. The topics can be related to molecular, physiological, behavioral, and other aspects of honeybees&rsquo and wild bees&rsquo health and extend to bee declines and pollination services.

Prof. Alberto Satta
Dr. Panagiotis Theodorou
Guest Editors

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Cannabis Extract As A Remedy for Dying Bees (Biology)

Bees are not able to protect themselves against pesticides and at huge chemical concentrations they die. Danel Załuski, Dr. habil. NCU professor from Collegium Medicum is a member of a team whose aim is to search for specimens of natural origin which protect insects.

“Help bees”, “Save bees”, “Save our bees”, “Be like Bee” are only some of online actions which are to protects our buzzing friends. What is all the fuss about? Albert Einstein was the one who already warned that “if bees disappear, human kind will soon follow their fate”, as the production of 75% of food in the world depends on the insects which pollinate flowers. And they are massively dying out. The reason is not only nosemosis or varroosis caused by excessive multiplications of the parasite Varroa destructor. Beekeepers are also concerned with the collapse of bee families outside beehives. Scientists have proved that the massive extinction of bees happens due to the use of insecticides on farm lands and in forests – insect-killing neonicotinoids, i.e., neuroactive substances from a pesticide group which disrupt bees’ orientation and communication.

Cannabis extract as a remedy for dying bees
fot. Nadesłane

Pesticides are chemical compounds, for instance derivatives of phenoxyacetic acid, organochlorine or organophosphate compounds, which are used mainly in agriculture and gardening, but also in forestry, veterinary sciences and textile materials impregnation. Apart from active substances, pesticides also contain emulsifiers, preservatives or other auxiliary substances which, for certain, are not indifferent to the environment. Spraying seeds with nicotinoids makes these compounds penetrate the whole plant, including flowers and pollen. Therefore, by collecting pollen and carrying it into the beehive, bees transport poison, which affects not only the insects working outside the beehive but also those which stay inside including the queen. – Pesticides usually affect bees’ nervous system, they change their behavior and decrease immunity – explains Daniel Załuski, Dr. habil, NCU professor, from the Department of Pharmaceutical Botany and Pharmacology of Collegium Medicum. – I have not heard of any substances which would protect bees against the negative influence of insecticides from the group of nicotinoids. Therefore, our research team have begun research on developing such substances of natural origin.

Dr hab. Daniel Załuski, prof. UMK fot. Nadesłane

There is a lot to protect bees against as pesticides may affect them on different layers. Contact substances affect their nervous system This may cause even chemicals which are considered to be nontoxic to poison a bee though direct contact. Pesticides may enter the bee’ organism with contaminated food, for example nectar, pollen, honeydew or water. Depending on the degree of toxicity of the formulation, bees may die instantly or in the beehive. Death in the beehive triggers off other consequences as the contaminated material poisons young bees and further contaminates honey with the pesticide. Bees do not have protective mechanisms against pesticides. At high concentration, they are defenseless.

Dr hab. Aneta Ptaszyńska, prof. UMCS fot. Archiwum NAWA/Alicja Szulc

Apart from professor Daniel Załuski, there are in the research team Aneta Ptaszyńska, Dr. habil, prof. of University of Maria Skłodowska-Curie in Lublin, and Rafał Kuźniewski, Dr., from the Department Pharmaceutical Botany and pharmacology of CM NCU. The problem of massive extinction of bees has been within the scientists’ interest since 2012. – As it often happens in science, the actions undertaken and commercialized initiatives came into life by accident – says professor Załuski. The main subject of my scientific activity are vegetable raw materials with adaptogenic affect, which increases the efficiency of a human organism through the influence on the immune, endocrine or nervous systems. Yet, professor Ptaszyńska has begun her research to seek natural substances which fight against nosemosis in honeybees. – Once, she told me about a tragic situation of bees and the possibility of their extinction, which causes my anxiety. We both agreed that it was worth looking for natural substances of plant origin which could stimulate the immune system of bees. The effect of many years of research is developing a formulation which fights against nosemosis in bees, and which has been on the market since 2018.

Dr Rafał Kuźniewski fot. Nadesłane

The cooperation has not ended with only one project. Scientists have begun research on plants like cannabis, which is the main subject of interest of doctor Kuźniewski. Unexpectedly it turned out that aquatic and ethanolic extracts increase survival of bees in conditions exposed to the presence of pesticides. The scientists obtained the essences from the leaf, stem and roots though the method of extraction supported with ultrasounds, and with application of water and water solutions of alcohols as solvents. Next, the extracts were lyophilizated and used in the tests which examined the survivability of bees in conditions stimulated with the extract and pesticide. The scientists solved the essence of cannabis in food, that is in high fructose corn syrup or a mixture of honey and powdered sugar.

The research model comprised 30 standard cages, each containing 40 bees. The bees were being given the extract for eight days, on the seventh day they were contaminated with imidacloprid or acetamiprid, and for two days they were given food with the extract. Survivability of the bees in particular cages was compared to survivability in the control sample, which was not stimulated with the formulation. It was also there that the bigger number of dead bees was recorded – arithmetic means calculated upon the three cages in the end of the experiment were respectively five and seven alive bees. The lowest number of dead bees was recorded in the groups which were provided with ethanolic or aquatic extract of cannabis leaves (Cannabis sativa L.) – the number of living bees in the end was 19 and 16.

Scientists find it relatively difficult to unequivocally answer the question if the cannabis extract may protect bees against a harmful impact of all pesticides or only selected ones.

Chemical differentiation of pesticides is huge, therefore environmental research should be done in the areas with highly developed agricultural production based on polycultures, which require application of various pesticides. – explains professor Załuski. – Such research usually takes two or three years, and it requires seasonal application of the mixture of food and extract to the bees. In our experiments, we usually used two most often applied pesticides from the group of neonicotinoids: imidacloprid and acetamiprid.

It turned out unexpectedly that the extracts of roots, leaves or stems of cannabis, which were obtained with aquatic solution of ethanol, both prolong the life of bees exposed to pesticides of nicotinoid group, and also decrease the amount of pathogenic spore of Nosema spp. This considerably limits nosemosis in bees.

In April 2020, the scientists made two patent applications, and currently they are preparing an application for an international patent. If they find a licensee, the product should enter the market in two or three years.


Results

Experiment 1: Acute exposure

The olfactory learning performance of 171 individual bees from 6 colonies was tested. Bees that extended their proboscis to fewer than 5 odour presentations when their antennae were touched were classed as unresponsive and excluded from further analyses (n = 29: Table S1), resulting in an average of 35.5 bees tested per treatment (34 in control, 37 in 250 ppb, 36 in 10 ppb and 35 in 2.4 ppb treatment groups). Pesticide treatment affected both the trainability and learning level of bees (Fig. 1, Table 1a). More bees were trainable to the conditioned odour in the control and 2.4 ppb groups compared to the 250 ppb treatment group (Fig. 1a). Control bees also displayed a higher learning level than those from both the 10 ppb and 250 ppb treatment groups (Fig. 1b, Table 1a). While there was no significant difference between control and 2.4 ppb groups, post-hoc comparisons revealed that 2.4 ppb treated bees showed a higher learning level than both the 250 ppb (Tukey, Z value = 5.694, p < 0.0001) and 10 ppb (Tukey, Z value = 3.479, p = 0.0028) groups (Fig. 1b). We found no difference in worker body size across treatment groups (linear mixed effects model, F3,164 = 0.28, p = 0.8396), although there was a significant effect of body size in some models as larger bees showed a higher overall learning level (Table 1).

Results from experiment 1: acute exposure.

(a) The mean proportion of bees in each acute treatment group that were trainable (trainability). (b) The mean number of conditioned responses of all acutely exposed bees per treatment group (learning level). (c) Acquisition curves showing the mean proportion of acutely exposed bees responding with a proboscis extension to the conditioned odour prior to reward over 15 conditioning trials. (d) Memory recall of the conditioned association (illustrated by mean proportion of bees that showed the conditioned response to the presented odour on trial 15 (dark grey bars) and 3 hours after the learning task in the memory test (light grey bars)) from trainable bees). Letters indicate significantly different pairwise comparisons from post-hoc tests (p < 0.05) and error bars indicate SE.

The learning ability of trainable bees (n = 78 bees in total: 23 bees in control, 24 in 2.4 ppb, 19 in 10 ppb and 12 in 250 ppb treatments) was not affected by treatment (Fig. 1). Control bees neither learned the task quicker (Table S2b), nor displayed the conditioned response more frequently (Fig. 1d), than the other treatment groups, with the average bee responding to the odour for the first time at trial 8 (Table S2, Electronic Supplementary Material (ESM)). Similarly, the performance of bees in the memory task was not significantly different after three hours compared to the end of the training period for any treatment group (compare dark and lighter grey columns in Fig. 1d, related samples Wilcoxon signed rank tests: 2.4 ppb p = 0.715 10 ppb p = 0.180 250 ppb p = 0.655 control p = 0.317), indicating there was no overall impact of acute pesticide exposure on memory performance.

Experiment 2: Chronic exposure

We tested the learning performance of 100 bees from 20 colonies (5 bees per colony), of which 5 unresponsive bees were removed from our analysis, resulting in 34 bees tested in control, 29 in 2.4 ppb and 32 in 10 ppb treatments (95 bees in total). We found no effect of pesticide exposure on either the number of bees that were trainable (Fig. 2a) or their learning level (Fig. 2b, Table S3). However, comparing only the performance of trainable bees (26 bees in control, 19 in 2.4 ppb and 19 in 10 ppb treatments: 64 bees in total), we found that control bees learnt the task faster than bees in both the 2.4 ppb (27% faster) and 10 ppb (38% faster) treatment groups (i.e. on average, the first response by control bees happened earlier in the experiment (mean = trial 6.9) than for pesticide treated bees, with average first responses at trial 8.7 for 2.4 ppb and 9.5 for 10 ppb (Table 2b), although final levels of task performance in terms of the proportion of bees responding to the conditioned odour and the learning level of these individuals was comparable across treatment groups after 15 trials (Fig. 2a, d, Table 2a)).

Results from experiment 2: chronic exposure.

(a) The mean proportion of bees in each chronic treatment group that were trainable (trainability). (b) The mean number of conditioned responses of all chronically exposed bees per treatment group (learning level). (c) Acquisition curves showing the mean proportion of chronically exposed bees responding with a proboscis extension to the conditioned odour prior to reward over 15 conditioning trials. (d) Memory recall of the conditioned association (illustrated by mean proportion of bees that showed the conditioned response to the presented odour on trial 15 (dark grey bars) and 3 hours after the learning task in the memory test (light grey bars)) from trainable bees). Letters indicate significant differences (p < 0.05) and error bars show SE.

The 3-hour period between the end of conditioning and the memory test had no significant impact on the proportion of control bees displaying conditioned responses to the odour (Related samples Wilcoxon signed ranked test, p = 0.317 Fig. 2d). However, the proportion of bees exposed to 2.4 ppb pesticide that showed the conditioned response to the odour was significantly lower after the 3 hour break compared to the end of the trial period (Related samples Wilcoxon signed ranked test, p = 0.027), showing an impact of pesticide on memory. Although the proportion of bees in the 10 ppb exposure group responding to the odour stimulus after 3 hours was lower than at the end of the trial period, this difference was not significant at the 5% level (Related samples Wilcoxon signed ranked test, p = 0.066). There were no significant differences in worker body size across treatment groups (Linear mixed effects model, F2,17 = 2.83, p = 0.0869, although there was a trend for 10 ppb treated bees to be smaller).


Professor suspects that hive collapses are caused by pesticides, which also could hurt human health

Lu has continued to investigate the possible links among neonicotinoids, bees, and human health, saying the honeybee is a good model organism for potential pesticide impact, as well as for potential effects across generations. Credit: Wikipedia

It's become something of a rite of spring. Every March, newspaper stories sprout about local beekeepers opening their hives to find an ongoing environmental mystery.

Instead of hungry bees ready for the first flights of spring, honeycombs that should be empty after a long winter are full, and instead the hives are empty. For some reason, during winter's coldest months, these bees chose to leave the hive to perish outside.

Colony collapse disorder, as the condition is known, remains a mystery with troubling implications for the fate of the human food supply, which depends, in part, on pollinators like the honeybee. Explanations that have been offered include pathogens, modern beekeeping practices, malnutrition, climate change, and pesticides.

It is that last possible cause that stands out to Harvard School of Public Health's (HSPH) Chengsheng (Alex) Lu, an associate professor of environmental exposure biology, who believes that the potential human health implications of colony collapse disorder extend beyond the drop in pollination—though that is worrisome enough—to the impact on humans of long exposure to low-level poisons like neonicotinoid pesticides, which have been suspected in the bee disorder. To Lu, it is an open question whether there are links between the pesticide and the recent increase in neurological conditions in children such as autism and ADHD.

To get to the bottom of the mystery, Lu has conducted pioneering research on the impact of neonicotinoid pesticides on honeybees. In a study published in 2012, he replicated colony collapse disorder experimentally, feeding bees sugar water with different levels of neonicotinoids over 13 weeks in the summer and watching what happened.

At first, nothing did. The hives seemed unaffected and healthy as they got ready for winter. Then, the week before Christmas, roughly three months after the neonicotinoid treatment was halted, hives began to fail. Eventually 15 of 16 hives collapsed, even those treated with the lowest dose.

The work was noted for providing a concrete link to neonicotinoids, which are the world's most widely used group of insecticides.

One particularly disturbing aspect of the work, which Lu described during a lunchtime "Hot Topics" talk on Aug. 12 at HSPH's Kresge Building, is that the bees that abandoned the hive during the collapse weren't the individuals that ate the sugar water laced with neonicotinoids. During summer's period of high activity, bees live just 35 days, so the colony that collapsed contained the next generation of bees, indicating that the effect may have been passed on between generations.

Lu has continued to investigate the possible links among neonicotinoids, bees, and human health, saying the honeybee is a good model organism for potential pesticide impact, as well as for potential effects across generations.

Neonicotinoids, chemicals similar to the nicotine produced by tobacco plants, have become widespread in part because of their ease of use, Lu said. Because they're water-soluble, the chemicals are taken up by a plant and spread throughout its tissues. Seed companies have made distribution even easier for farmers by coating seeds with the chemical, which ensures the plants sprouting from them contain the pesticide.

The chemicals are present not just in food plants, but are also widely represented in nursery stock, including plants sold at major garden retailers, Lu said. They're also found in the environment, and Lu said there are questions about their role in the loss of birds and aquatic invertebrates.

Lu described it as a race against time to save the bees, which are routinely transported around the country by commercial beekeepers to pollinate agricultural fields. He spoke to one blueberry farmer who said that before colony collapse disorder struck, he would pay $250,000 to have his fields pollinated. Today that figure stands at $750,000, and the cost is passed on to consumers.

Lu believes that the pesticide is fed to bees by unsuspecting beekeepers. The pesticide is widely used on corn, which is used to make high-fructose corn syrup. The corn syrup is mixed with water and routinely fed to bees by commercial beekeepers.

Affected bees, which include wild honeybees, Lu said, exhibit a range of neurological conditions, including disorientation, flying back to the wrong colonies, and abandoning colonies in winter.

"The [phrase] 'bee-line' is no longer valid," Lu said. "The question … is do these things also apply to human health?"


Acknowledgements

We thank Kate Vaughan-Williams for help with data collection Dara Stanley, Gemma Baron, Callum Martin, Mark Brown and Aaishah Manan for advice and technical assistance and Dave Goulson for providing worker size distribution data. E.L.’s contribution was partly funded by a Leverhulme Trust Early Career Fellowship. This study was funded by a London BBSRC DTP studentship to E.E.W.S. R.J.G.’s research is supported by the Grand Challenges in Ecosystems and the Environment Initiative at Silwood Park.