Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees Christopher Moffat, Stephen T. Buckland, Andrew J. Samson, Robin McArthur, Victor Chamosa Pino, Karen A. Bollan, Jeffrey T.-J. Huang & Christopher N. Connolly
Scientific Reports 6, Article number: 24764 (2016) doi:10.1038/srep24764
There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.
Insect pollinators are essential pollinators of many native wildflowers and agriculturally important crops. Although pesticides are widely used to protect our crops, they are also exposed to many beneficial insects and may be contributing to the worldwide decline in pollinators and other beneficial insects1,2. The economic value of insect pollination is around US$215 billion (2005)3, as pollination improves crop yield, quality, shelf life and commercial value4,5,6. The neonicotinoids are a widely used class of insecticides that are nicotine-based compounds often used as systemic insecticides (seed coating) on bee-visited crops such as oilseed rape (canola) and ornamental garden plants or as foliar sprays on top fruit such as apples and pears. Once absorbed into the plant following seed germination, neonicotinoids can translocate throughout the plant, contaminating (at low ppb) their nectar and pollen during crop flowering7. The crop nectar and pollen may be consumed by bees, or transported back to the nest to feed their larvae or for long-term storage. As neonicotinoids persist in the soil8, they may also compromise the function of soil organisms that contribute to soil fertility and so limit crop yield9. Once in the soil they can translocate to wildflowers in field margins10,11 and so prolong exposure to insect pollinators, well beyond crop flowering12 and even into the following year8.
Neonicotinoids act as nicotinic acetylcholine receptor (nAChR) agonists, which provide the majority of the excitatory neurotransmission in the insect central nervous system. Imidacloprid (IMD) is a partial agonist13, evoking 10–14% of maximal ACh-induced responses14 in Kenyon cells. However, thiamethoxam (THX) has been found to be an inactive pro-pesticide that lacks functional activity on isolated cells15,16, although high affinity binding sites have been reported in some17, but not all18 isolated membrane preparations. Thiamethoxam may be metabolized to clothianidin (CLO) or desmethyl thiamethoxam18, both of which are neuroactive, stimulating receptor responses15. In contrast, CLO is classed as a super agonist, evoking larger currents than ACh14. The neonicotinoids act predominantly on Kenyon cells that are the major neuronal cell type in the mushroom bodies of the bee brain19. These mushroom bodies are higher order brain structures mediating multisensory integration, learning and memory20. Given that field relevant levels21,22 of IMD and CLO have been shown to activate nAChRs in Kenyon cells19,21, it is anticipated that these compounds would influence cognitive function in bees so long as they can reach the brain at neuroactive levels following oral exposure (ie. nectar and pollen consumption). Field levels of neonicotinoids are generally accepted to be between 1–10 ppb22, but are not acutely lethal to bees at this level. Nevertheless, evidence on bumblebees (Bombus terrestris audax), indicates that neuroactive levels (~10 nM, 2.5 ppb) can reach their brain within 3 days of dietary exposure to 2.5 ppb IMD21. This is consistent with evidence that neonicotinoids have been shown to exert a negative impact on higher cognitive tasks in bees such as olfactory learning & memory23, foraging24,25, navigation26, queen fidelity27,28 and whole colony performance21,24,29, but see30. Neonicotinoids also disrupt the sex ratio in parasitic wasps31,32, solitary bees33 and possibly honeybees34.
The term neonicotinoid is commonly used in preference to the name of the actual compound under study in the belief that they are all essentially identical in their targets and effects. However, individual neonicotinoids, IMD, THX and CLO have been reported to exhibit distinct binding to nicotinic acetylcholine receptors35 and so may exert distinct effects on, and pose differential risks to bees. Indeed, as with most pharmacologically active compounds, small changes to the ligand, or the receptor binding site, may cause dramatic differences in their affinity for specific receptors. In this respect, our knowledge of the risks from neonicotinoids has been generated largely from chronic studies on IMD21,25, a cocktail of THX and CLO27,33 or THX and IMD34. In contrast, our knowledge on CLO, when present alone, is limited to its acute exposure effects7,10, under which conditions there is no apparent risk at field relevant levels. In the case of field trials, exposure to other known33,34,36 or unknown pesticides, disease or mitigating factors30 may occur, confounding interpretation. In this study, we compared all three EU-banned neonicotinoids, IMD, THX and CLO, for effects on bumblebees (Bombus terrestris audax) to determine whether they act consistently and in predictable ways, where CLO would be expected to be the most toxic, given its higher potency and THX requiring metabolism to CLO to exert an identical toxic effect.
The first level at which differential effects of neonicotinoids could occur is by variability in their ability to reach their site of action, the bee brain. We demonstrated previously that bumblebees exposed to 10 nM (2.1 ppb w/w) IMD in their diet (sugar syrup) accumulated, within 3 days, between 4 and 10 nM within their brains21. Here, we demonstrate using stable isotope dilution liquid chromatography-mass spectrometry that dietary exposure to CLO (2.5 ppb) or THX (2.5 ppb), for 3 days, also delivers CLO (14.7 ± 2.0 and 3.9 ± 0.6 nM, respectively) to the brain (mean ± S.E.M, Fig. 1A), with levels of CLO in the hemolymph being 3.4 ± 0.6 (CLO fed bees) and 0.5 ± 0.1 nM (THX fed bees) respectively (Fig. 1A). Thiamethoxam levels in brains and hemolymph from THX fed bees were below the limit of quantification (Supplementary Figure S1).
In humans, chronic nicotine exposure leads to an upregulation of high-sensitivity (to nicotine and acetylcholine) receptors38 that is postulated to drive reward pathways and so contribute to the addictive actions of nicotine39. Similarly, the upregulation of human receptors can be induced by neonicotinoid exposure35, albeit at the high doses (μM) required for their receptor binding. In honeybees, neonicotinoid-seeking behavior is induced by exposure to IMD or THX, but not CLO40. Therefore, we investigated in bees whether a chronic exposure to field-relevant levels of neonicotinoids (2.5 ppb IMD, THX or CLO for 7 days) might increase their sensitivity to subsequent exposure to toxic levels of CLO as suggested previously41. A
The target site for the neonicotinoids is the nicotinic acetylcholine receptors (nAChRs) commonly expressed in mushroom bodies, higher order insect brain structures that mediate multisensory integration, learning & memory19. Differential nAChR subtype activation by each neonicotinoid could explain the variability seen for acute toxicity, anti-feedant effects and increasing sensitivity to toxic levels of CLO (see also whole colony effects presented later). To investigate this, Kenyon cell neurons in culture were exposed sequentially to IMD (10 nM), CLO (10 nM) and then acetylcholine (1 mM) and receptor responses monitored by Ca2+ influx using Fura-2AM. Both IMD and CLO generate acute Ca2+ responses that desensitized (Ca2+ response returns to baseline) in the continued presence of the neonicotinoid. On the basis of a cell’s responsiveness to field-relevant levels of IMD, CLO and ACh, we determined that there are 4 distinct cell populations of Kenyon cells in our cultures, those responding to IMD only, CLO only, those responding to both IMD and CLO, or those responding to ACh only. All neurons were activated by acetylcholine (Fig. 1D) as expected for Kenyon cells. Although IMD-activated receptors are desensitized and unable to respond to subsequent activation by IMD (for example see top trace, Fig. 1D), other cells still demonstrated responses to CLO despite a prior exposure to IMD (second and third traces, Fig. 1D). In particular, in the third trace, we can see that CLO can activate receptors despite the evidence that IMD-responsive receptors have desensitized. Therefore, at a field relevant dose, IMD and CLO clearly activate distinct nAChRs and different Kenyon cell populations. Therefore, it is possible that differential receptor/Kenyon cell activation may underlie the differential effects identified here and also indicates that they may exert different risks to bumblebees and other insect pollinators.
Given these differential neonicotinoid effects, we investigated whether there is also a differential risk to bumblebee colonies in the field. Colonies were exposed to neonicotinoids (provided at 2.5 ppb in sugar syrup) over 5 weeks. Bees had full outside access after 1 day (a delay to allow transportation to site), ensuring that bees were not forced to consume the neonicotinoids as in laboratory experiments. To ensure that the relevance of our findings were not restricted to a single habitat type or period of season, we investigated the performance of 75 colonies (producing 5884 bees, 5365 brood and 727 queens) using 5 distinct sites across Scotland over 5 separate overlapping periods during the summer of 2015. Three of these sites were located within (Fife and Perth), or close to (Dundee), highly intensive arable agriculture, one site was in a mixed livestock/natural habitat (Aberfeldy) and one was in a remote, relatively pristine site in the Highlands of Scotland (Kyle of Lochalsh). To demonstrate the performance of all colonies, scatter plots of colony performance (n = 18 for each condition presented) illustrate the number of live bees, viable brood, number of queens (see supplementary Figure S3 for determination) and the normalized change in nest mass for each treatment (Fig. 2A–D).
Growing evidence of the sublethal impact of field-relevant exposure to imidacloprid on honeybees indicates deficits on neuronal Kenyon cells function in the mushroom bodies19, which are major sites of learning and memory and multisensory integration. This neuronal dysfunction provides mechanistic insight into the evidence on honeybees for the impairment of olfactory learning and memory23,43, motor control44 and navigation26. In the case of THX, it has been demonstrated to impair olfactory memory43, motor control44 and, when exposed in combination with CLO, colony performance and queen survival27. In contrast to these negative impacts of THX, field studies conducted by Syngenta30,45 have reported no negative impact. However, the study and interpretations of Pilling30 have been contested46 and the other study45 lacked replication, with two control sites but only a single treatment site used. Therefore, further evidence is required to resolve the risk to honeybees from THX. One possible explanation for the discrepancy between laboratory and field trials is that honeybee colonies are very large and may be able to buffer the negative effects of chemical toxicity. Indeed, THX (or IMD that was found as an unexpected contaminant) has been shown to be detrimental to honeybees, causing increasing mortality over time34. However, the honeybee colonies appeared to compensate for the loss of workers by decreasing their production of non-working males (drones). In contrast to the mixed effects reported for IMD and THX, no effects of CLO, either alone47, or in combination with β–cyfluthrin36 were reported on honeybee colonies, suggesting that the effects reported by Sandrock27 may be due to a direct action of THX48 or its metabolic products18,49. Therefore, no clear evidence exists to implicate field-relevant exposure to CLO in whole honeybee colony losses.
For bumblebees, the evidence is more robust and consistent, with IMD being reported to impair bumblebee pollen foraging efficiency24,25 and colony impairment21,24,29. Similarly, exposure to THX caused bees to learn more slowly and impaired their short-term memory50. As for IMD, THX exposure resulted in poorer foraging and can consequentially have a negative impact on crops, with bumblebees exposed to THX providing diminished crop pollination services51. In contrast, in a field trial at a single site for THX-treated OSR (22 colonies), no negative impacts were reported on colony growth45, although no assessment of live bees remaining was possible as nests were frozen prior to assessment. In the case of clothianidin, B. terrestris colony growth was observed to be decreased in colonies associated with seed coated (clothiandin and β–cyfluthrin) oilseed rape, but given that a field-relevant mixture was used, the causative agent is unclear36.
A difficulty in assessing the field relevance of studies is that laboratory studies often do not provide a natural choice of forage and so the dose of pesticide delivered may exceed that encountered in the environment. In contrast, field studies limit exposure of bees to the field conditions under study (usually the flowering period of a crop) to minimize exposure to unknown confounding factors such as other pesticides. However, this also creates an artificial situation, with bees on site for a short duration. This has 2 problems. Firstly, a prolonged neonicotinoid exposure by its translocation to wildflowers8,10,11,12 is excluded from the study. Secondly, the expected deficit in bees is subtle, affecting their learning and memory19,23,43 and so their foraging ability24,25,51, which may not be adequately tested in a pristine habitat with (presumably) abundant and diverse wildflower availability that is lacking in a typical arable habitat.
Given the shortfalls of both laboratory and field studies in terms of field-relevance, we conducted a semi-field trial where neonicotinoids were provided as an optional supply of sugar syrup, but the bees were free to forage and needed to gather their own pollen in order to grow and raise brood. In addition, the sites selected covered a range of different habitats, from intensive arable to managed wilderness. No obvious differences between the relative effects of the neonicotinoids were observed between sites (although the sample size for between site analyses was small).
A prerequisite for the toxicity of neonicotinoids on insects is their access to nAChRs that are restricted to the brain. We demonstrate here, that all three neonicotinoids, when exposed at field relevant levels (2.5 ppb), can reach the brain in an active form at 4–15 nM (~1–4 ppb) after 3 days dietary exposure. None of the neonicotinoids are lethal to bees at field-relevant (1–10 ppb) exposure levels22, whereas exposure to 100 ppb causes mortality for CLO (91%) and THX (71%), but not IMD, indicating a toxicity order of CLO > THX > IMD. However, all IMD exposed (100 ppb) bees were immobile from day 1, while many CLO/THX exposed bees remained highly active. As IMD exposed bees were immobile, they do not feed37 and so exposure limits toxicity at high doses. Regardless, no anti-feedant effects are evident at field relevant levels and consequences from chronic exposure are possible.
Distinct consequences of particular receptor subtype activation, or as a result of differential cell stimulation may underlie the differences in acute toxicity observed and may also drive selective long-term changes following chronic exposure. Although IMD is acutely non-lethal to individual bees (at 100 ppb), it is chronically toxic to whole colonies (at 2.5 ppb), but exposure to field-relevant levels (2.5 ppb) fails to increase sensitivity to subsequent CLO exposure. THX is acutely toxic to bees and also chronically toxic to whole colonies (at 2.5 ppb). In addition, THX also alters the sex ratio in the nest and field-relevant levels increase sensitivity to subsequent exposure to high level CLO. Surprisingly, despite the acute toxicity of CLO and the ability of field-relevant levels (2.5 ppb) to increase sensitivity to high level CLO, we find no evidence for any negative effects on bumblebee colonies following a chronic exposure to CLO at field realistic levels. In fact, CLO-treated colonies produced more queens suggesting a possible beneficial impact.
To understand the potential difference between acute and chronic exposure to neonicotinoids, it may be relevant to consider the analogous situation in man, where prolonged low level exposure to nicotine occurs during smoking (and presumably vaping). Chronic nicotine administration by human smokers causes an upregulation of nicotine binding sites in the cerebral cortex38, a region involved in cognition, attention and memory. Indeed, acute exposure to nicotine improves attention, learning and memory. In humans, the major brain subtype is the α4β2 receptors, which are preferentially upregulated by low (nM) levels of nicotine to favor the high sensitivity stoichiometry (α4)2(β2)3 over low sensitivity (α4)3(β2)2 receptors. In addition, receptors may undergo a conformational change that results in higher affinity receptors52. The consequential pharmacological upregulation of function is postulated to drive reward pathways and so contribute to the addictive actions of nicotine in man39,53.
Interestingly, IMD, thiacloprid and particular metabolites have also been shown to upregulate (5–8 fold) mammalian α4β2 receptors with a potency of 500–70,000 nM41. In bumblebees, where neonicotinoids are more potent, we showed previously that sensitivity of nAChR was enhanced following chronic exposure to IMD21 supporting the case for an upregulation of receptor function. Such a mechanism is consistent with the neonicotinoid-seeking behavior reported in honeybees following exposure to IMD and THX, but not CLO40.
Our findings that an increased vulnerability to CLO results from exposure to field relevant levels of either CLO or THX indicates that receptor upregulation also results from chronic exposure to CLO and possibly from THX metabolites (THX does not bind to receptors). The failure of IMD to increase vulnerability to CLO appears inconsistent, unless IMD activates distinct receptors from CLO. The differential effects on individual bees and whole colonies may be the consequence of different receptor subtype function, or cell-specific roles, that may be differentially activated by individual neonicotinoids. We confirmed this potential using Kenyon cells in culture exposed sequentially to IMD, CLO and ACh. We find that most cells responded to only IMD or CLO, with few responding to both. Therefore, neonicotinoids display different cellular profiles that may drive distinct behavioral phenotypes.
The differential effect of THX on the sex bias of surviving bees in a colony is not observed for CLO (or IMD) suggesting that THX may also operate independently of its CLO metabolite. This is supported by the reported side effects of THX on the midgut of honeybees54 and its selective effect on grooming44 and may be explained by the novel metabolites produced by THX, but not CLO, such as desmethyl-THX. Desmethyl-THX has been reported to have a 28- to 3600-fold increased affinity for nAChRs over THX15,18,55. However, THX has also been reported to have a high binding affinity, albeit to a different site or receptor state56. Alternatively, it has been reported that THX and desmethyl-THX may generate formaldehyde in some species49 that causes protein-DNA crosslinking and chronic toxicity. Interestingly, an alteration in sex ratio by neonicotinoids has been reported previously in solitary bees33, honeybees34 and parasitic wasps31,32 and may result directly by influencing sex allocation31 or indirectly by possible selective loss of adult workers (female)34 or female pupae32.
In terms of differential toxicity to IMD and THX, both have been reported to impair honeybee23 and bumblebee50 learning and memory and both stimulate neonicotinoid-seeking behavior40. Together, these behavioral changes may underlie colony dysfunction reported here and elsewhere21,24,29. In contrast to IMD and THX, CLO does not appear to be detrimental to bumblebee colonies.
Our data supports the recent conclusion17 that there is no single conserved site for neoncitoinoid binding, but several sites with distinct binding pockets that is dependent on receptor composition. We postulate that the cells and/or specific receptor subtypes activated by CLO may not control learning and memory or drive neonicotinoid preference seeking behavior in B. terrestris and so does not disrupt individual bee or whole colony performance when exposed chronically at field relevant levels. It is important to bear in mind that small changes in neonicotinoid structure may have a major impact on its affinity and receptor selectivity, leading to differences in its acute and/or chronic toxicity. Equally, small changes in receptor structure, as occurs between species, presumably underlie their differential sensitivity to neonicotinoids. It is important to emphasize that the differential toxicity of IMD and THX, but not CLO, to bumblebees, may be a species-specific profile that is unique to B. terrestris. Therefore, we propose that the findings on individual neonicotinoids are not extrapolated to other neonicotinoids and that findings in a particular species are not extrapolated to other species.
How to cite this article: Moffat, C. et al. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees. Sci. Rep. 6, 24764; doi: 10.1038/srep24764 (2016).