
==== Front
PeerJ
PeerJ
PeerJ
PeerJ
2167-8359
PeerJ Inc. San Diego, USA

17978
10.7717/peerj.17978
Entomology
Potential acetylcholine-based communication in honeybee haemocytes and its modulation by a neonicotinoid insecticide
Pamminger Tobias 12
Basley Kate 1
http://orcid.org/0000-0003-4421-2876
Goulson Dave 1
Hughes William O. H. 1william.hughes@sussex.ac.uk

1 School of Life Sciences, University of Sussex, Brighton, UK
2 Bayer AG, Monheim am Rhein, Germany
Cutler G. Christopher
13 9 2024
2024
12 e179783 3 2018
6 8 2024
© 2024 Pamminger et al.
2024
Pamminger et al.
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

There is growing concern that some managed and wild insect pollinator populations are in decline, potentially threatening biodiversity and sustainable food production on a global scale. In recent years, there has been increasing evidence that sub-lethal exposure to neurotoxic, neonicotinoid pesticides can negatively affect pollinator immunocompetence and could amplify the effects of diseases, likely contributing to pollinator declines. However, a direct pathway connecting neonicotinoids and immune functions remains elusive. In this study we show that haemocytes and non-neural tissues of the honeybee Apis mellifera express the building blocks of the nicotinic acetylcholine receptors that are the target of neonicotinoids. In addition, we demonstrate that the haemocytes, which form the cellular arm of the innate immune system, actively express choline acetyltransferase, a key enzyme necessary to synthesize acetylcholine. In a last step, we show that the expression of this key enzyme is affected by field-realistic doses of clothianidin, a widely used neonicotinoid. These results support a potential mechanistic framework to explain the effects of sub-lethal doses of neonicotinoids on the immune function of pollinators.

Haemocytes
Pesticide
Innate immune system
Immune regulation
Clothianidin
Neonicotinoid
Bee health
Immunosuppression
EC FP7 Marie Curie FellowshipPIEF-GA-2013-626585 Tobias Pamminger was funded by an EC FP7 Marie Curie Fellowship PIEF-GA-2013-626585. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Body
pmcIntroduction

Pollinating insects such as bumblebees are of major ecological and economic importance, but many of their populations are in decline (Potts et al., 2010; Vanbergen et al., 2013). Threats include natural diseases and emerging diseases related to the globalized pollinator-trade that have negative effects on both managed and wild pollinator populations (Fürst et al., 2014; Goulson & Hughes, 2015; McMahon et al., 2015; Wilfert et al., 2016). While healthy pollinator communities are sometimes able to cope with such diseases, additional stressors can compromise pollinator immunity, potentially resulting in lethal epidemics (Botías et al., 2021; Goulson et al., 2015). One factor that is now known to impact pollinator immunity is their exposure to sub-lethal doses of pesticides. Many studies have now demonstrated that exposure to neurotoxic neonicotinoids in particular, can significantly impair multiple components of the cellular and humoral immune response, with consequent effects on parasite replication (Annoscia et al., 2020; Brandt et al., 2016; Di Prisco et al., 2013; López et al., 2017; Malladi et al., 2023; Orčić et al., 2022). While the detrimental effects of neurotoxic pesticides on pollinator behaviour and navigation are intuitive (Fischer et al., 2014; Henry et al., 2012; Jin et al., 2015; Stanley et al., 2016), the strong immunosuppressive effects of these neurotoxic pesticides are difficult to mechanistically explain (Sanchez-Bayo et al., 2016). The close ontogenetic connection between haemocytes and the nervous system has been proposed as a possible explanation (Pamminger et al., 2018), with haemocytes having been shown to express receptors to biogenic amine neurotransmitters for example (Huang et al., 2012; Qi et al., 2016), but this mechanism remains to be investigated.

In vertebrates it is well established that the immune system has a close regulatory connection with the nervous system (Sternberg, 2006). In particular, the ancient cholinergic signalling system based on acetylcholine (ACh) has been demonstrated to perform a pivotal role in maintaining homeostasis of the immune system (Kawashima et al., 2012; Sternberg, 2001). Evidence for a functionally similar ACh-based immune regulatory network has more recently emerged in several bivalve mollusc, crustacean and insect species (Chen et al., 2016; Giordani et al., 2023; Shi et al., 2014, 2012; Zhang et al., 2021). In particular, haemocytes, the cellular arm of the invertebrate immune system, have been demonstrated in oysters to not only express subunits of the muscarinic (mAChR) and nicotinic acetylcholine receptors (nAChR), but also to directly respond to the presence of ACh (Chen et al., 2015; Liu et al., 2016b). In insects, haemocytes have also been found to express and have receptors for nAChR and ACh, with knockdown of Ach synthesis in Drosophila haemocytes reducing expression of a gene for antimicrobial peptide production (Giordani et al., 2023; Xu et al., 2017). Since neonicotinoids, and other insecticides, target nAChR receptors with high affinity (Christen & Fent, 2017; Tomizawa & Casida, 2003), the presence of a neural-independent, ACh-based communication system in the innate immune system of pollinators could provide a direct mechanistic link for immunosuppression by neonicotinoids and other insecticides (Pamminger et al., 2018).

In this study, we investigate if non-neural immune-relevant tissues (fatbody, midgut and haemocytes) of the honeybee Apis mellifera: 1) express nAChR subunits and choline acetyltransferase (ChAT), a key enzyme to synthesize ACh, and 2) if such a system can respond to a field-realistic dose of neonicotinoid that bees could encounter under natural conditions.

Methods

Portions of this text were previously published as part of a preprint (https://doi.org/10.1101/105700).

Bee collection

Foraging Apis mellifera worker were collected between July and September 2016 from a single colony on the campus of the University of Sussex, Brighton, UK (50°52′02.8″N 0°05′09.6″W). We used foragers because they have mature immune systems and are the bees most directly exposed to neonicotinoids on flowers. In all cases bees were collected between 09:00 and 11:00 in order to minimize gene expression variation caused by circadian rhythms. They were placed in 50 mL Falcon tubes with three bees per tube. The tubes contained a moist cotton ball as a water source and to regulate relative humidity within the tube.

Experiment 1: tissue-specific expression of nAChR subunits and ChAT

The bees collected for Experiment 1 (tissue expression levels) were directly put on ice to cold anaesthetise them. After cold immobilization (~10 min) the bees were decapitated using a sterile razor blade, dissected under RNA Later (Thermo Fisher Scientific, Waltham, MA, USA) using a sterile dissection kit and either whole brain (N = 5), fatbody (N = 7) or midgut (N = 7) was extracted (one type of tissue per bee). For haemolymph extraction, the thorax and abdomen of the bees were carefully punctured after decapitation using a sterile dissection needle and haemolymph was collected using a sterile graded glass capillary. The haemolymph of two bees was pooled (total 16 bees; N = 8) and haemocytes were collected following standard protocol (Negri et al., 2014). All tissues were homogenized in Trizol (ABI, New York, NY, USA) using a sterile pestle and total RNA was extracted following the manufacturer’s instructions. The concentration and purity of RNA was determined on a Nanodrop 2000®.

Experiment 2: clothianidin exposure

To test whether neonicotinoid exposure affects ChAT expression, sixty-two foraging A. mellifera workers were collected and randomly assigned to either treatment (N = 30) or control (N = 32). After collection, the bees were placed in their Falcon tubes in a dark incubator at 33 °C and 80% relative humidity. The tubes had a hole drilled in one end through which the bees were provided with 60% sucrose solution via 10 mL syringe feeders. The bees were kept at these conditions for 20 h for acclimatisation before the start of the experiment. Following the 20 h acclimatisation period, the feeders were removed. A total of 4 h later the treatment group was provided with new feeders containing 60% sucrose solution spiked with 5 ppb clothianidin (using molecular grade acetone as solvent), while the control received sucrose solution with the same concentration of acetone only. The clothianidin concentration was chosen as a field-realistic exposure level, within the range of residue levels reported for treated crops (Botias et al., 2015; Sanchez-Bayo & Goka, 2014), and resulted in bees ingesting a dose that was approximately an order of magnitude less than the oral LD50 of 3.8 ng/bee (Bartling, Vilcinskas & Lee, 2019; European Commission, 2005). All feeders were weighed before and after the experiment to the closest 0.001 g using a Kern PFB 300-3 scale to measure the dose (ng) of neonicotinoids that the bees had consumed. All bees had access to the feeders for 24 h after which haemolymph was collected from all surviving individuals, and samples of two bees were pooled (resulting in N = 6 treatment, N = 7 control) following the procedure of Experiment 1.

Gene expression analysis

We quantified expression levels for the nine α (1-9) and two β (1-2) subunits of nAChR (Jones et al., 2006), and ChAT. We compared these to the reference gene rp49 (Lourenço et al., 2008). We used 100 ng of total RNA for reverse transcription using the Phusion RT-PCR kit (Thermo Fisher Scientific, Waltham, MA, USA). The purity of the RNA samples was checked using a Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) based on absorbance curve and the 260/280 and 260/230 ratios. Primers for all the target genes were designed using Primer3 (Untergasser et al., 2012) and published sequences available from GenBank (see Table S3 for details). Primer efficiencies were measured using a dilution series of Apis mellifera brain cDNA (pooled subsamples of five individuals) covering three orders of magnitude including the cDNA concentration used in the reaction. Primer efficiencies were found to be above 91% for all primer pairs. Reaction specificity was confirmed by melting curve analysis. All analyses were performed on a StepOnePlus™ Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) using SYBR green assays and were analysed using the StepOne software.

Gene expression analysis of the nAChR subunits α1-9, β1-2, ChAT and rp49 were performed in 10 μL reactions using GoTaq® qPCR Master Mix (Promega, Madison, WI, USA) and 0.5 μM of each primer (Sigma-Aldrich, St. Louis, MO, USA) on the StepOnePlus™ Real-Time PCR System. Samples of cDNA corresponding to 2 ng total RNA in 2 μL volumes were added and each sample analysed in three technical replicates. Each plate contained one negative control reaction for each primer pair using pooled and 1:10 diluted RNA extracts from five randomly chosen individuals in order to control for gDNA contamination. The following program was used for amplification: 95 °C for 2 min, followed by 40 cycles of 30 s of 95 °C denaturation, 30 s annealing at 59 °C and 30 s extension at 72 °C following by a melting curve to ensure PCR specificity. The data used for the analysis were the target gene expression normalized to the rp49 reference gene expression using averages of the technical replicates and the 2−ΔΔCT method (Rao et al., 2013). Note that although the rp49 reference gene shows good expression stability across honeybee tissues (Lourenço et al., 2008), the use of only a single reference gene means that comparisons in absolute expression levels between tissues should be interpreted cautiously.

Data analysis

To compare the nAChR subunit expression patterns we used the programme PRIMER 6, version 6.1.13, + add-in, version 1.0.3 (PRIMER-E Ltd) to perform permutational multivariate analysis of variance (PERMANOVA) with the normalized relative expression of all 11 subunits as the response and tissue as the predictor variable. PERMANOVA is free of assumptions about variable distributions (Anderson, 2017). All tests were carried out using 9,999 permutations on a resemblance matrix using Chad distance estimates and the robustness of the results were tested using the Euclidian distance as an alternative estimate. We performed a SIMPER analysis to compare the expression of individual nAChR subunits according to tissue identity and tissue differentiation. All other tests were performed in R 3.2.4 (R Development Core Team, 2015). Survival was analysed as the proportion of bees that died over the duration of the experiment using a GLM with binomial data distribution. The other results of Experiment 2 were analysed using non-parametric statistics (Kruskal-Wallis and Wilcoxon tests) and Bonferroni corrections in cases of multiple testing. The MDS plot was generated in PRIMER 6; all other graphs were done in R using the sciplot package (Morales, 2011).

Results

All the investigated tissues (fat body, haemocyte, midgut, brain) expressed nAChR, with the relative expression pattern of the different subunits differing between tissues (Pseudo-F3,23 = 7.76, P < 0.001; Figs. 1A–1D). The expression patterns of the subunits differed significantly between all four tissues in pairwise comparisons (t > 1.92 and P < 0.006 in all cases; Table S1). In the brain, α7 was very highly expressed compared to the other subunits, with α2 and, to a lesser extent, α5 and β1 being moderately expressed relative to the remaining subunits (Fig. 1D). Expression in the haemocytes was also highest for α2 and α7, with α8, α9, β1 and β2 being higher than the remaining subunits (Fig 1B). Expression in the fatbody was dominated by α9 and β2 (Fig. 1A), while expression in the midgut was relatively low and similar for most of the subunits (Fig. 1C). Brain, midgut and, to a lesser extent, fatbody samples formed distinct clusters in multidimensional scaling plot, with the haemocyte samples occupying a larger area that overlapped partially with fatbody (Fig. 2). The SIMPER analysis indicates that the largest differences between tissues, in terms of the expression pattern of the different subunits, were between the brain and the other tissues, with the very high expression of α7 in the brain relative to other subunits being a consistent cause of this (Table S2). We also found evidence of expression of ACh, measured as choline acetyltransferase (ChAT) expression, with the expression being very high in the brain samples, lower in the haemocytes and very low in the midgut and fatbody (Fig. 3). The expression levels of ChAT differed significantly between the tissues (χ2 = 21.96, P < 0.001; though note this could also relate to differences between tissues in expression of the reference gene).

10.7717/peerj.17978/fig-1 Figure 1 Mean ± s.e. relative expression of the nAChR subunits α1-9 and β1-2 in honeybees.

Expression values were normalized against rp49. Data are for fatbody (A in red, N = 7); haemocytes (B in blue, N = 8); midgut (C in white, N = 7); brain (D in green, N = 5; note the much higher expression of a7 indicated by the break in the y-axis).

10.7717/peerj.17978/fig-2 Figure 2 Multidimensional scaling (MDS) plot of nAChR expression in honeybees.

Plot is based on Chad distance. Data are for brain (green crosses, N = 5), haemocytes (blue inverted triangles, N = 8), fatbody (red triangles, N = 7) and midgut (black circles, N = 7).

10.7717/peerj.17978/fig-3 Figure 3 Mean ± s.e. relative expression of the A. mellifera choline transferase gene (ChAT) in honeybees.

Data are for brain (green, B, N = 5); haemocytes (blue, H, N = 8); fatbody (red, F, N = 7); midgut (white, M, N = 7). The inset shows the role of ChAT in acetylcholine synthesis. Only brain and haemocyte cells exhibit robust ChAT expression. Different letters above columns indicate significant expression differences between cell types.

In Experiment 2, Treatment and Control bees consumed similar amounts of sucrose solution (mean ± s.e. 0.064 ± 0.001 and 0.06 ± 0.001 mL per bee respectively, W = 114, P = 0.95). This equated to a consumption of 0.3 ± 0.008 ng of clothianidin by the Treatment bees. Treatment and Control bees did not differ in survival (z = −0.26, P = 0.79), with mortality being relatively high in both cases (16/32 bees and 16/30 bees, respectively). Importantly, we found that ChAT expression was significantly increased in the haemocytes of bees exposed to clothianidin (W = 38, P = 0.014), with the expression levels in bees treated with clothianidin being almost 2.5 times higher than in Control bees, Fig. 4).

10.7717/peerj.17978/fig-4 Figure 4 The effect of clothianidin on choline transferase (ChAT) expression in honeybee haemocytes.

Graph shows the mean ± SE relative expression of ChAT normalised against the rp49 reference gene. Bees were either treated with the neonicotinoid clothianidin (red; N = 6) or control (white; N = 7). Asterisks above the columns indicate that expression in clothianidin and control bees differed significantly.

Discussion

In this study we demonstrate the widespread expression of nAChR subunits in non-neural and immune-relevant tissues in the honeybee A. mellifera. In addition, we show that haemocytes in A. mellifera express the key enzyme to synthesize ACh, which suggests that in principal all components for an ACh-based communication (receptor and signalling molecule) are expressed. Lastly, we experimentally establish that sub-lethal, field realistic doses of the neonicotinoid clothianidin can influence the expression pattern of the ChAT communication system in vivo.

Our results are in line with recent findings, which suggest the presence of an non-neural and immune-related ACh-based communication in a range of invertebrates (Chen et al., 2015; Giordani et al., 2023; Liu et al., 2016b; Shi et al., 2014; Zhang et al., 2021). Similar to our findings, different combinations of nAChR subunits have been found to be expressed in a wide range of non-neural tissues in a lepidopteran insect (Xu et al., 2017). In both our results and those of Xu et al. (2017), α7 was the dominant subunit in the brain, α9 and β2 were the most highly expressed subunits in the fatbody, and all subunits had very low expression in the midgut. We found α2 and α7 to be the most highly expressed subunits in honeybee haemocytes, whereas Xu et al. (2017) found α3 to be the most highly expressed subunit in the lepidopteran haemocytes, with α2, α7 and the other subunits having similarly low expression. The expression of these sub-units by itself does not automatically indicate the presence of functional receptors (Aztiria, Sogayar & Barrantes, 2000). However, the fact that haemocytes can respond to the presence of ACh in molluscs suggests that, at least in some species, functional receptors are most likely present (Liu et al., 2016b; Shi et al., 2014). In addition, haemocytes have been shown to synthesize acetylcholine-degrading enzymes (acetylcholinesterase) in scallops, likely terminating ACh-based haemocyte excitation following pathogen exposure (Shi et al., 2012), and expression of the nAchR subunit α7 in haemocytes has been shown to be necessary for production of an important antimicrobial peptide in Drosophila fruit flies (Giordani et al., 2023). Our results are in keeping with these findings and indicate additionally that haemocytes in principal may express the enzymatic machinery to actively synthesize ACh themselves. Taken together these lines of evidence suggest that invertebrate innate immune systems may possess all the essential components for sending, receiving and terminating ACh based signals. It is consequently possible that, similarly to their vertebrate counterparts (Kawashima et al., 2012), the invertebrate innate immune system utilizes ACh-based communication.

Subunits of nAChR subunits were also expressed by secondary immune-relevant tissues, the fatbody and the midgut (Xing et al., 2021; Zhu et al., 2022). This was similarly the case in a lepidopteran stem borer (Xu et al., 2017), with expression of all subunits being similarly low in the midgut, while α9 and β2 were relatively highly expressed compared to other subunits in the fatbody. It would be interesting to investigate whether haemocytes could utilize ACh-based signals to convey information to the fatbody and midgut, thereby coordinating the systemic immune response during infections.

In addition to establishing that honeybee haemocytes express nAChR subunits and ChAT, we found that exposure to the neonicotinoid clothianidin affected ChAT expression in the haemocytes of honeybees. We found this effect at a dose of 0.3 ng/bee, which is approximately an order of magnitude less than the LD50 of 3.8 ng/bee (Bartling, Vilcinskas & Lee, 2019; European Commission, 2005; Lewis et al., 2016). It has been experimentally shown that clothianidin at 10 ppb negatively affects the encapsulation, melanisation and antimicrobial immune properties of haemolymph in honeybees, with other neonicotinoids causing similar effects (Annoscia et al., 2020; Brandt et al., 2016). Our finding of an effect on gene expression of clothianidin at 5 ppb is in keeping with this. The effect we observed could have been an indirect effect, for example from clothianidin inducing detoxification pathways, but it is in keeping with the direct effect of the neonicotinoid on ACh signalling in haemocytes that would be predicted by the haemocytes having nAChR receptors. The fact that clothianidin increased expression of ChAT could suggest that it will produce an increase in the production of antimicrobial peptides and therefore resistance to disease (Giordani et al., 2023; Hanson & Lemaitre, 2023). Low levels of stress, including from pesticides, can result in increased gene expression and stimulatory effects on a diversity of biological functions (Rix & Cutler, 2022). However, this would be contrary to the immunosuppressive effects of clothianidin and other neonicotinoids that have been abundantly demonstrated (Annoscia et al., 2020; Brandt et al., 2016; Di Prisco et al., 2013; López et al., 2017; Malladi et al., 2023; Orčić et al., 2022). An alternative explanation is that the clothianidin induces overstimulation of the nAChR receptors or off-target synthesis of choline acetyltransferase and acetylcholine that negatively impacts cell function and homeostasis. Neonicotinoid insecticides are designed to target nAChR receptors with high affinity (Elbert et al., 2008; Matsuda et al., 2001), causing lethal effects through receptor overstimulation (Tomizawa & Casida, 2003; Tomizawa, Lee & Casida, 2000). In molluscs, the blocking of haemocyte-based mAChR before pathogen challenge promotes the expression of Tumor Necrosis Factor (TNF), which in turn results in elevated haemocyte apoptosis (Liu et al., 2016a, 2016b). If a similar, nAChR-based, regulatory connection is present in the haemocytes of pollinators, nAChR blockage by neonicotinoids could directly explain their detrimental effects on haemocytes and by extension the immunosuppressive effects observed in honeybees (Brandt et al., 2017; Di Prisco et al., 2013; Malladi et al., 2023). In addition to confirming the presence of functional receptors and ACh communication in the non-neural tissues of bees, future work should investigate the effects of neonicotinoid exposure on expression of the nAChR subunits and other components of the acetylcholine signalling machinery in haemocytes, and the downstream impacts of these effects. Our results suggest that haemocytes may use different receptor subunits than the brain, so determining the relative sensitivity of haemocytes to neonicotinoids compared with other tissues also warrants further investigation.

While the direct effects of neonicotinoids on neuronally-associated traits such as behaviour, memory and navigation are intuitive (Blacquière et al., 2012; Fischer et al., 2014; Jin et al., 2015), the effects on other traits such as immunity or reproduction have not previously been adequately explained (Straub et al., 2016; Whitehorn et al., 2012; Williams et al., 2015). The finding that non-neural tissues including haemocytes can potentially express nAChR could explain these counterintuitive effects by providing a mechanism for direct interaction with these tissues (Pamminger et al., 2018). These systemic pesticides migrate into both pollen and nectar, so pollinators are exposed to them when visiting treated crops or contaminated wildflowers (Botias et al., 2015; Goulson et al., 2015). Once ingested, the pesticide is absorbed via the gut and passes through the haemolymph on the way to its designated target sites in the central nervous system (Tomizawa & Casida, 2003). In the haemolymph, neonicotinoids inevitably come into contact with haemocytes, with potentially disruptive effects for haemocyte function if haemocytes are sensitive to neonicotinoids. In addition, the differences between tissues in the relative expression patterns of nAChR subunits could help to explain the pronounced differences in susceptibility to neonicotinoids between different developmental stages, species and experiments (Grewal, Power & Shetlar, 2001; Moffat et al., 2016; Tomizawa & Casida, 2003; Whitehorn et al., 2012). Since the subunit composition determines the binding properties and consequently toxicity of neonicotinoids, and such composition varies between species, tissues, life stages and time, this variation could explain the observed differences in toxicity by orders of magnitude (Govind, Vezina & Green, 2009; Moffat et al., 2016; Tomizawa & Casida, 2003, 2009; Whitehorn et al., 2012; Xu et al., 2017).

Conclusions

In summary, our results support a mechanistically informed framework to understand the numerous unexplained side effects associated with sub-lethal neurotoxic pesticides exposure in pollinators. Such an analysis framework is urgently needed in order to identify and ultimately limit the numerous side effects of neurotoxic pesticides.

Supplemental Information

10.7717/peerj.17978/supp-1 Supplemental Information 1 Supplemental Tables.

10.7717/peerj.17978/supp-2 Supplemental Information 2 Raw data.

We thank all members of the Hughes lab and three anonymous reviewers for their useful comments on previous versions of the MS.

Additional Information and Declarations

Competing Interests

Author Contributions

Data Availability

The authors declare that they have no competing interests. Tobias Pamminger is employed by Bayer AG.

Tobias Pamminger conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Kate Basley performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Dave Goulson conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

William O. H. Hughes conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

The following information was supplied regarding data availability:

The raw data is available in the Supplemental File.
==== Refs
References

Anderson (2017) Anderson MJ Balakrishnan N Colton T Everitt B Piegorsch W Ruggeri F Teugels JL Permutational multivariate analysis of variance (PERMANOVA) Wiley StatsRef: Statistics Reference Online 2017 1 15 10.1002/9781118445112.stat07841
Annoscia et al. (2020) Annoscia D Di Prisco G Becchimanzi A Caprio E Frizzera D Linguadoca A Nazzi F Pennacchio F Neonicotinoid clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation Nature Communications 2020 11 5887 10.1038/s41467-020-19715-8
Aztiria, Sogayar & Barrantes (2000) Aztiria EM Sogayar MC Barrantes FJ Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems Neurochemical Research 2000 25 171 180 10.1023/a:1007512121082 10685617
Bartling, Vilcinskas & Lee (2019) Bartling MT Vilcinskas A Lee KZ Sub-lethal doses of clothianidin inhibit the conditioning and biosensory abilities of the western honeybee Apis mellifera Insects 2019 10 340 10.3390/insects10100340 31614672
Blacquière et al. (2012) Blacquière T Smagghe G van Gestel CAM Mommaerts V Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment Ecotoxicology 2012 21 973 992 10.1007/s10646-012-0863-x 22350105
Botias et al. (2015) Botias C David A Horwood J Abdul-Sada A Nicholls E Hill E Goulson D Neonicotinoid residues in wildflowers, a potential route of chronic exposure for bees Environmental Science & Technology 2015 49 12731 12740 10.1021/acs.est.5b03459 26439915
Botías et al. (2021) Botías C Jones JC Pamminger T Bartomeus I Hughes WOH Goulson D Multiple stressors interact to impair the performance of bumblebee Bombus terrestris colonies Journal of Animal Ecology 2021 90 415 431 10.1111/1365-2656.13375 33084067
Brandt et al. (2016) Brandt A Gorenflo A Siede R Meixner M Büchler R The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.) Journal of Insect Physiology 2016 86 40 47 10.1016/j.jinsphys.2016.01.001 26776096
Brandt et al. (2017) Brandt A Grikscheit K Siede R Grosse R Meixner MD Büchler R Immunosuppression in honeybee queens by the neonicotinoids thiacloprid and clothianidin Scientific Reports 2017 7 4673 10.1038/s41598-017-04734-1 28680118
Chen et al. (2015) Chen H Wang LL Zhou Z Hou ZH Liu ZQ Wang WL Gao DH Gao Q Wang MQ Song LS The comprehensive immunomodulation of NeurimmiRs in haemocytes of oyster Crassostrea gigas after acetylcholine and norepinephrine stimulation BMC Genomics 2015 16 942 10.1186/s12864-015-2150-8 26576764
Chen et al. (2016) Chen H Zhou Z Wang LL Wang H Liu R Zhang H Song LS An invertebrate-specific miRNA targeted the ancient cholinergic neuroendocrine system of oyster Open Biology 2016 6 160059 10.1098/rsob.160059 27488375
Christen & Fent (2017) Christen V Fent K Exposure of honey bees (Apis mellifera) to different classes of insecticides exhibit distinct molecular effect patterns at concentrations that mimic environmental contamination Environmental Pollution 2017 226 48 59 10.1016/j.envpol.2017.04.003 28402838
Di Prisco et al. (2013) Di Prisco G Cavaliere V Annoscia D Varricchio P Caprio E Nazzi F Gargiulo G Pennacchio F Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees Proceedings of the National Academy of Sciences of the United States of America 2013 110 18466 18471 10.1073/pnas.1314923110 24145453
Elbert et al. (2008) Elbert A Haas M Springer B Thielert W Nauen R Applied aspects of neonicotinoid uses in crop protection Pest Management Science 2008 64 1099 1105 10.1002/ps.1616 18561166
European Commission (2005) European Commission Unit D.3-chemicals, contaminants and pesticides: clothianidin Directorate-General HCP, editor 2005 Brussels European Commission
Fischer et al. (2014) Fischer J Müller T Spatz AK Greggers U Grünewald B Menzel R Neonicotinoids interfere with specific components of navigation in honeybees PLOS ONE 2014 9 e91364 10.1371/journal.pone.0091364 24646521
Fürst et al. (2014) Fürst MA McMahon DP Osborne JL Paxton RJ Brown MJF Disease associations between honeybees and bumblebees as a threat to wild pollinators Nature 2014 506 364 366 10.1038/nature12977 24553241
Giordani et al. (2023) Giordani G Cattabriga G Becchimanzi A Di Lelio I De Leva G Gigliotti S Pennacchio F Gargiulo G Cavaliere V Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity Insect Biochemistry and Molecular Biology 2023 153 103899 10.1016/j.ibmb.2022.103899 36596348
Goulson & Hughes (2015) Goulson D Hughes WOH Mitigating the anthropogenic spread of bee parasites to protect wild pollinators Biological Conservation 2015 191 10 19 10.1016/j.biocon.2015.06.023
Goulson et al. (2015) Goulson D Nicholls E Botías C Rotheray EL Bee declines driven by combined stress from parasites, pesticides, and lack of flowers Science 2015 347 1255957 10.1126/science.1255957 25721506
Govind, Vezina & Green (2009) Govind AP Vezina P Green WN Nicotine-induced upregulation of nicotinic receptors: underlying mechanisms and relevance to nicotine addiction Biochemical Pharmacology 2009 78 756 765 10.1016/j.bcp.2009.06.011 19540212
Grewal, Power & Shetlar (2001) Grewal PS Power KT Shetlar DJ Neonicotinoid insecticides alter diapause behavior and survival of overwintering white grubs (Coleoptera: Scarabaeidae) Pest Management Science 2001 57 852 857 10.1002/ps.373 11561413
Hanson & Lemaitre (2023) Hanson MA Lemaitre B Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis Disease Models & Mechanisms 2023 16 dmm049965 10.1242/dmm.049965 36847474
Henry et al. (2012) Henry M Béguin M Requier F Rollin O Odoux J-F Aupinel P Aptel J Tchamitchian S Decourtye A A common pesticide decreases foraging success and survival in honey bees Science 2012 336 348 350 10.1126/science.1215039 22461498
Huang et al. (2012) Huang J Wu SF Li XH Adamo SA Ye GY The characterization of a concentration-sensitive α-adrenergic-like octopamine receptor found on insect immune cells and its possible role in mediating stress hormone effects on immune function Brain Behavior and Immunity 2012 26 942 950 10.1016/j.bbi.2012.04.007 22561607
Jin et al. (2015) Jin NX Klein S Leimig F Bischoff G Menzel R The neonicotinoid clothianidin interferes with navigation of the solitary bee Osmia cornuta in a laboratory test Journal of Experimental Biology 2015 218 2821 2825 10.1242/jeb.123612 26206356
Jones et al. (2006) Jones AK Raymond-Delpech V Thany SH Gauthier M Sattelle DB The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera Genome Research 2006 16 1422 1430 10.1101/gr.4549206 17065616
Kawashima et al. (2012) Kawashima K Fujii T Moriwaki Y Misawa H Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function Life Sciences 2012 91 1027 1032 10.1016/j.lfs.2012.05.006 22659391
Lewis et al. (2016) Lewis KA Tzilivakis J Warner DJ Green A An international database for pesticide risk assessments and management Human and Ecological Risk Assessment: An International Journal 2016 22 1050 1064 10.1080/10807039.2015.1133242
Liu et al. (2016a) Liu ZQ Wang LL Zhou Z Sun Y Wang MQ Wang H Hou ZH Gao DH Gao Q Song LS The simple neuroendocrine-immune regulatory network in oyster Crassostrea gigas mediates complex functions Scientific Reports 2016a 6 26396 10.1038/srep26396 27193598
Liu et al. (2016b) Liu ZQ Zhou Z Wang LL Dong WJ Qiu LM Song LS The cholinergic immune regulation mediated by a novel muscarinic acetylcholine receptor through TNF pathway in oyster Crassostrea gigas Developmental and Comparative Immunology 2016b 65 139 148 10.1016/j.dci.2016.07.003 27394930
López et al. (2017) López JH Krainer S Engert A Schuehly W Riessberger-Gallé U Crailsheim K Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae Scientific Reports 2017 7 40853 10.1038/srep40853 28145462
Lourenço et al. (2008) Lourenço AP Mackert A dos Santos Cristino A Simões ZLP Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR Apidologie 2008 39 372 385 10.1051/apido:2008015
Malladi et al. (2023) Malladi S Sukkar D Bonnefoy A Falla-Angel J Laval-Gilly P Imidacloprid and acetamiprid synergistically downregulate spaetzle and myD88 of the Toll pathway in haemocytes of the European honeybee (Apis mellifera) Environmental Toxicology and Pharmacology 2023 104 104323 10.1016/j.etap.2023.104323 37995888
Matsuda et al. (2001) Matsuda K Buckingham SD Kleier D Rauh JJ Grauso M Sattelle DB Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors Trends in Pharmacological Sciences 2001 22 573 580 10.1016/s0165-6147(00)01820-4 11698101
McMahon et al. (2015) McMahon DP Fürst MA Caspar J Theodorou P Brown MJF Paxton RJ A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees Journal of Animal Ecology 2015 84 615 624 10.1111/1365-2656.12345 25646973
Moffat et al. (2016) Moffat C Buckland ST Samson AJ McArthur R Pino VC Bollan KA Huang JTJ Connolly CN Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees Scientific Reports 2016 6 24764 10.1038/srep24764 27124107
Morales (2011) Morales M Sciplot: scientific graphing functions for factorial designs 2011 R package version 1.2-0 https://github.com/mutualism/sciplot
Negri et al. (2014) Negri P Maggi M Szawarski N Lamattina L Eguaras M Apis mellifera haemocytes in-vitro: what type of cells are they? Functional analysis before and after pupal metamorphosis Journal of Apicultural Research 2014 53 576 589 10.3896/IBRA.1.53.5.11
Orčić et al. (2022) Orčić SM Čelić TV Purać JS Vukašinović EL Kojić DK Acute toxicity of sublethal concentrations of thiacloprid and clothianidin to immune response and oxidative status of honey bees Apidologie 2022 53 50 10.1007/s13592-022-00959-w
Pamminger et al. (2018) Pamminger T Botías C Goulson D Hughes WOH A mechanistic framework to explain the immunosuppressive effects of neurotoxic pesticides on bees Functional Ecology 2018 32 1921 1930 10.1111/1365-2435.13119
Potts et al. (2010) Potts SG Biesmeijer JC Kremen C Neumann P Schweiger O Kunin WE Global pollinator declines: trends, impacts and drivers Trends in Ecology & Evolution 2010 25 345 353 10.1016/j.tree.2010.01.007 20188434
Qi et al. (2016) Qi YX Huang J Li MQ Wu YS Xia RY Ye GY Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors Elife 2016 5 e12241 10.7554/eLife.12241 26974346
R Development Core Team (2015) R Development Core Team R: a language and environment for statistical computing 2015 Vienna, Austria R Foundation for Statistical Computing
Rao et al. (2013) Rao X Huang X Zhou Z Lin X An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis Biostatistics, Bioinformatics and Biomathematics 2013 3 3 71 85 25558171
Rix & Cutler (2022) Rix RR Cutler GC Review of molecular and biochemical responses during stress induced stimulation and hormesis in insects Science of the Total Environment 2022 827 154085 10.1016/j.scitotenv.2022.154085 35218848
Sanchez-Bayo & Goka (2014) Sanchez-Bayo F Goka K Pesticide residues and bees–a risk assessment PLOS ONE 2014 9 e94482 10.1371/journal.pone.0094482 24718419
Sanchez-Bayo et al. (2016) Sanchez-Bayo F Goulson D Pennacchio F Nazzi F Goka K Desneux N Are bee diseases linked to pesticides?-A brief review Environment International 2016 89–90 7 11 10.1016/j.envint.2016.01.009
Shi et al. (2014) Shi XW Wang LL Zhou Z Liu R Li YC Song LS Acetylcholine modulates the immune response in Zhikong scallop Chlamys farreri Fish & Shellfish Immunology 2014 38 204 210 10.1016/j.fsi.2014.03.008 24680755
Shi et al. (2012) Shi XW Zhou Z Wang LL Yue F Wang MQ Yang CY Song LS The immunomodulation of acetylcholinesterase in Zhikong scallop Chlamys farreri PLOS ONE 2012 7 e30828 10.1371/journal.pone.0030828 22292052
Stanley et al. (2016) Stanley DA Russell AL Morrison SJ Rogers C Raine NE Investigating the impacts of field-realistic exposure to a neonicotinoid pesticide on bumblebee foraging, homing ability and colony growth Journal of Applied Ecology 2016 53 1440 1449 10.1111/1365-2664.12689 27867216
Sternberg (2001) Sternberg E Neuroendocrine regulation of autoimmune/inflammatory disease Journal of Endocrinology 2001 169 429 435 10.1677/joe.0.1690429 11375112
Sternberg (2006) Sternberg EM Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens Nature Reviews Immunology 2006 6 318 328 10.1038/nri1810
Straub et al. (2016) Straub L Villamar-Bouza L Bruckner S Chantawannakul P Gauthier L Khongphinitbunjong K Retschnig G Troxler A Vidondo B Neumann P Williams GR Neonicotinoid insecticides can serve as inadvertent insect contraceptives Proceedings of the Royal Society of London B 2016 283 20160506 10.1098/rspb.2016.0506
Tomizawa & Casida (2003) Tomizawa M Casida JE Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors Annual Review of Entomology 2003 48 339 364 10.1146/annurev.ento.48.091801.112731
Tomizawa & Casida (2009) Tomizawa M Casida JE Molecular recognition of neonicotinoid insecticides: the determinants of life or death Accounts of Chemical Research 2009 42 2 260 269 10.1021/ar800131p 19053239
Tomizawa, Lee & Casida (2000) Tomizawa M Lee DL Casida JE Neonicotinoid insecticides: molecular features conferring selectivity for insect versus mammalian nicotinic receptors Journal of Agricultural and Food Chemistry 2000 48 12 6016 6024 10.1021/jf000873c 11312774
Untergasser et al. (2012) Untergasser A Cutcutache I Koressaar T Ye J Faircloth BC Remm M Rozen SG Primer3-new capabilities and interfaces Nucleic Acids Research 2012 40 15 e115 10.1093/nar/gks596 22730293
Vanbergen et al. (2013) Vanbergen AJ Baude M Biesmeijer JC Britton NF Brown MJF Brown M Bryden J Budge GE Bull JC Carvel C Challinor AJ Connolly CN Evans DJ Feil EJ Garratt MP Greco MK Heard MS Jansen VAA Keeling MJ Kunis WE Marris GC Memmott J Murray JT Nicolson SW Osborne JL Paxton RJ Pirk CWW Polce C Potts SG Priest NK Raine NE Roberts S Ryabov EV Shafir S Shirley MDF Simpson SJ Stevenson PC Stone GN Termansen M Wright GA Insect Pollinators I Threats to an ecosystem service: pressures on pollinators Frontiers in Ecology and the Environment 2013 11 5 251 259 10.1890/120126
Whitehorn et al. (2012) Whitehorn PR O’Connor S Wackers FL Goulson D Neonicotinoid pesticide reduces bumble bee colony growth and queen production Science 2012 336 351 352 10.1126/science.1215025 22461500
Wilfert et al. (2016) Wilfert L Long G Leggett HC Schmid-Hempel P Butlin R Martin SJM Boots M Deformed wing virus is a recent global epidemic in honeybees driven by Varroa mites Science 2016 351 594 597 10.1126/science.aac9976 26912700
Williams et al. (2015) Williams GR Troxler A Retschnig G Roth K Yañez O Shutler D Neumann P Gauthier L Neonicotinoid pesticides severely affect honey bee queens Scientific Reports 2015 5 14621 10.1038/srep14621 26459072
Xing et al. (2021) Xing WH Zhou DD Long Q Sun MH Guo R Wang LM Immune response of Eastern honeybee worker to Nosema ceranae infection revealed by transcriptomic investigation Insects 2021 12 728 10.3390/insects12080728 34442293
Xu et al. (2017) Xu G Wu SF Teng ZW Yao HW Fang Q Huang J Ye GY Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae) Insect Science 2017 24 371 384 10.1111/1744-7917.12324 26847606
Zhang et al. (2021) Zhang X Pan LQ Tong RX Li YF Tian YM Li DY Si LJ PacBio full length transcript sequencing and Illumina transcriptome insight into immune defense mechanism of Litopenaeus vannamei under ammonia-N stress Aquaculture 2021 536 736457 10.3390/ijms231911474
Zhu et al. (2022) Zhu ZW Wang J Fan XX Long Q Chen HZ Ye YP Zhang KY Ren ZM Zhang Y Niu QS Chen DF Guo R CircRNA-regulated immune responses of asian honey bee workers to microsporidian infection Frontiers in Genetics 2022 13 1013239 10.3389/fgene.2022.1013239 36267412
