Concentration-dependent effects of acute and chronic neonicotinoid exposure on the behaviour and development of the nematode Caenorhabditis elegans
2017; Wiley; Volume: 73; Issue: 7 Linguagem: Inglês
10.1002/ps.4564
ISSN1526-4998
AutoresMonika M Kudelska, Lindy Holden‐Dye, Vincent O’Connor, D. Doyle,
Tópico(s)Insect and Arachnid Ecology and Behavior
ResumoPest Management ScienceVolume 73, Issue 7 p. 1345-1351 Research ArticleFree Access Concentration-dependent effects of acute and chronic neonicotinoid exposure on the behaviour and development of the nematode Caenorhabditis elegans Monika M Kudelska, Corresponding Author Monika M Kudelska mk11g11@soton.ac.uk Biological Sciences, University of Southampton, Southampton, UKCorrespondence to: MM Kudelska, Biological Sciences, University of Southampton, Southampton, UK. E-mail: mk11g11@soton.ac.ukSearch for more papers by this authorLindy Holden-Dye, Lindy Holden-Dye Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this authorVincent O'Connor, Vincent O'Connor Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this authorDeclan A Doyle, Declan A Doyle Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this author Monika M Kudelska, Corresponding Author Monika M Kudelska mk11g11@soton.ac.uk Biological Sciences, University of Southampton, Southampton, UKCorrespondence to: MM Kudelska, Biological Sciences, University of Southampton, Southampton, UK. E-mail: mk11g11@soton.ac.ukSearch for more papers by this authorLindy Holden-Dye, Lindy Holden-Dye Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this authorVincent O'Connor, Vincent O'Connor Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this authorDeclan A Doyle, Declan A Doyle Biological Sciences, University of Southampton, Southampton, UKSearch for more papers by this author First published: 06 March 2017 https://doi.org/10.1002/ps.4564Citations: 12AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract BACKGROUND Neonicotinoid insecticides are under review owing to emerging toxicity to non-target species. Interest has focused on biological pollinators while their effects on other organisms that are key contributors to the ecosystem remain largely unknown. To advance this, we have tested the effects of representatives of three major classes of neonicotinoids, thiacloprid, clothianidin and nitenpyram, on the free-living nematode Caenorhabditis elegans (C. elegans), as a representative of the Nematoda, an ecologically important phylum contributing to biomass. RESULTS Concentrations that are several-fold higher than those with effects against target species had limited impact on locomotor function. However, increased potency was observed in a mutant with a hyperpermeable cuticle, which shows that drug access limits the effects of the neonicotinoids in C. elegans. Thiacloprid was most potent (EC50 714 μm). In addition, it selectively delayed larval development in wild-type worms at 1 mm. CONCLUSION C. elegans is less susceptible to neonicotinoids than target species of pest insect. We discuss an approach in which this defined low sensitivity may be exploited by heterologous expression of insect nicotinic acetylcholine receptors from both pest and beneficial insects in transgenic C. elegans with increased cuticle permeability to provide a whole organism assay for species-dependent neonicotinoid effects. © 2017 Society of Chemical Industry 1 INTRODUCTION Since launching into the agrochemical market in the 1990s, neonicotinoids have become the most commonly used insecticides worldwide.1 Currently, there are six compounds used as crop protectants and one used as an external pest control agent. These are classified into N-nitroguanidine (imidacloprid, dinotefuran, thiamethoxam and clothianidin), N-cyanoamidines (acetamiprid and thiacloprid) and nitromethylenes (nitenpyram). Several factors contribute to their success, including high potency against a wide range of piercing and sucking pests,2 selective toxicity to insects over humans,3 distinct mode of action and advantageous psychochemical properties, which allow a diversity of application methods.4 Nevertheless, there are emerging issues associated with their use that currently focus on their potential toxicity towards one of the most important biological pollinators, bees.5 This focus has instigated many studies, but the effects on other biomass important organisms, e.g. other pollinators, molluscs6 or nematodes, have not been investigated in depth or systematically. Pharmacological and electrophysiological data suggest that nicotinic acetylcholine receptors (nAChRs) are the principal site of action of neonicotinoids.7-15 However, different neonicotinoids exhibit a differential mode of action, depending on the subtype of receptor investigated. Some act as full, some as partial and some as superagonists.16-19 As an example, clothianidin is a superagonist on hybrid chicken–Drosophila receptors16 and on the native nAChRs expressed in Drosophila CNS.17 In contrast, it acts as a partial agonist on cockroach CNS neurons.18 The widespread use of and issues associated with neonicotinoids require better understanding of their specificity, but even compounds with the same chemical pharmacophore will target distinct nAChR subunits.14, 20 An important limitation relates to the difficulty in achieving heterologous expression of insect nAChRs.21 The desire routinely to express these receptors has led to a search for cofactors that underpin their expression in vivo that can be transplanted to heterologous systems. An important step forward involved cross-referencing understanding of the molecular determinants of C. elegans receptor expression to the investigation of insect receptors. Thus, studies on the determinants of nAChR expression in C. elegans in vivo identified a nematode molecular chaperone RIC-3.22, 23 Its conserved role is shown by the observation that it is obligatory to expression of insect receptors in Xenopus oocytes.24 Therefore, transgenic approaches in the model organism C. elegans could serve as a platform to identify neonicotinoid-sensitive insect nAChRs. The C. elegans genome contains a predicted 29 nAChR subunits.25 A subset of these are expressed at the neuromuscular junction of the pharyngeal and the body wall muscle and are involved in feeding, locomotion and reproduction. In addition, they have wide expression in sensory and integrating circuits implicated in higher-function chemosensory-driven behavioural responses and other environmentally cued behaviours underpinning plasticity.26 This involvement of nAChRs in cholinergic pathways that regulate environmentally driven behavioural adaptation in C. elegans is similar to their role in pollinating insects, where it has been shown that neonicotinoids have the ability to disrupt neural plasticity.27-29 In the present study we have evaluated the impact of different chemical classes of neonicotinoids on C. elegans behaviours with a longer-term goal of establishing this as a transgenic model for the expression of insect nAChRs and characterisation of their interaction with neonicotinoids. We investigated the effects of three classes of neonicotinoids, thiacloprid, clothianidin and nitenpyram, and compared them with the definitive nAChR agonist nicotine. We exposed worms to high concentrations of drugs acutely (4 h) and chronically (24 h) and determined effects on locomotion and development. We indicate how the whole-organism approaches are facilitated by mutants that enable drug dosing in the intact organism. This identifies an ability to report acute and chronic effects of these important compounds and a potential route to investigate their ecotoxicology. This work provides a benchmark against which the genetic tractability of the worm can be used to investigate neonicotinoid mode of action. 2 METHODS 2.1 C. elegans maintenance Wild-type (N2 Bristol strain) and strain CB7431 [genotype: bus-17, (br2) X, outcrossed 4 times; obtained from CGC] C. elegans were cultured at 20 °C on nematode growth medium (NGM) agar plates seeded with 50 μL lawn of OP50 Escherichia coli strain.30 Experiments were performed on young hermaphrodite adults (L4 + 1 day). Drugs and reagents were purchased from Sigma Aldrich (Dorset, England). Observations were made using a binocular microscope, unless otherwise stated. Results are expressed as mean ± SEM of N determinations. Graph generation and measurement of EC50 were performed in GraphPad Prism v.6.07 (GraphPad Software Inc., La Jolla, CA). 2.2 Drug solutions Thiacloprid and clothianidin were dissolved in 100% dimethyl sulphoxide (DMSO) and added to experiments so the drug vehicle concentration was 0.5% v/v. Nitenpyram and nicotine stocks were prepared by dissolving drugs in dH2O and diluted to the indicated final concentrations. Thiacloprid, clothianidin and nicotine stocks were kept at −18 °C for up to 1 month, whereas nitenpyram stock was made immediately prior to experiments. Nitenpyram-containing solutions were protected from light between measurements to prevent photodegradation. 2.3 Acute exposure and effects on motility; thrashing assay In liquid, C. elegans make rhythmic stereotypical flexing movements centred on the midpoint of their body, called 'thrashing'. To assess the impact of neonicotinoids on C. elegans motility, thrashing was assayed. Experiments were performed in a 24-well plate in a final volume of 500 μL of phosphate buffer (M9)30 with 0.1% bovine serum albumin, with either drug/solvent or solvent alone as control. Typically, two (vehicle control) or six (treatment) worms were picked into a single well containing phosphate buffer and left for at least 5 min to equilibrate to the solution. Then thrashes, defined by a single inflection back and forth, were counted for a duration of 30 s, defined as time zero, to provide baseline measurement for each worm. Subsequently, either drug/solvent or solvent alone (50 μL) was added to the wells and carefully mixed by pipetting the bath volume up and down to give the final desired concentration. Thrashing rate was scored for 30 s at time points: 10, 30, 40, 60, 120 and 240 min post-addition of the drug/drug vehicle. 2.4 Chronic exposure and effects on motility; body bends To allow chronic exposure to drugs, experiments were performed in six-well plates on solid NGM agar in which three of the wells were seeded with E. coli OP50 to allow 24 h exposure of the young adult worm to the drug in the presence of food, and the remaining three wells were left E. coli free to provide an assay arena for motility. Clothianidin, thiacloprid and nicotine were incorporated into the medium by first making stock solutions in DMSO. Stock solutions of DMSO were added to molten NGM (55 °C) to give the final indicated concentration in which DMSO (vehicle) did not exceed 0.5%. Each well of the 6-well plate was filled with 3 mL of molten NGM either with vehicle control or with drug/vehicle and left to solidify. A quantity of 50 μL of E. coli OP50 (OD600 nm = 0.8–1) was added to three of the wells, and the plates were left in the fume hood for 2 h to dry. The remaining three wells were left without OP50. One-day-old adult hermaphrodites were placed in each of the wells containing OP50 with either drug/vehicle or vehicle only. After 24 h, worms were transferred into the wells without OP50 containing either drug/vehicle or vehicle only. After a 10 min acclimatisation period, the motility of worms on solid medium was measured by counting the number of body bends per minute. 2.5 Development assay One-day-old adult hermaphrodites (6–10) were placed into the wells of a six-well plate where the wells contained NGM, with or without drug or solvent, and were seeded with OP50, as described above. After 1 h they were removed from the wells, and the eggs they had laid were left behind. The larval development of hatched progeny was monitored. Developmental stages were identified following size/vulva/eggs present in the uterus according to criteria described by Karmacharya et al.31 L1 were the smallest worms on the plate. L2 were slightly bigger, L3 bigger still, more mobile and displaying a prevulva space. L4 were identified by the appearance of the premature vulva, whereas gravid adults had a fully formed mature vulva and eggs present in their uterus. These observations were typically made at 60× magnification, but when necessary, worms were viewed at higher magnification, under a Nikon Eclipse E800 microscope, at different time points: 24, 30, 48, 54, 72, 80, 96, 120 and 144 h after eggs were laid. Results are represented as worms in each developmental stage as a mean percentage of the total population size. 3 RESULTS 3.1 Nicotine and neonicotinoid effects on C. elegans motility Wild-type N2 worms can sustain rhythmic thrashing in liquid for 4 h, and this is subject to a concentration-dependent inhibition in the presence of nicotine (Fig. 1a). At the highest concentration tested, 100 mm, the inhibition is relatively rapid, and complete inhibition of thrashing is observed at 10 min. Solubility restricted the concentrations that could be tested for thiacloprid and clothianidin to a maximum of 1 and 2.5 mm, respectively, and neither of these compounds inhibited thrashing at these concentrations, in contrast to 1 mm of nicotine (Fig. 1a). Nitenpyram elicited an inhibition at 100 mm, but unlike nicotine this was not a complete paralysis. Figure 1Open in figure viewerPowerPoint The concentration dependence and time course for the inhibitory effect of nicotinic compounds on motility of C. elegans. Wild-type (a) and bus-17 (b) worms were acutely exposed to varying concentrations of nicotine, thiacloprid, clothianidin, nitenpyram or drug vehicle (control) in a liquid medium. Their effect on thrashing over time was recorded. Data are the mean ± SEM collected over at least two observations, with the number of determinations specified in brackets. One-way ANOVA (Kruskal–Wallis test) with Dunn's corrections: * P ≤ 0.05, ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. 3.2 Effect of nicotine and neonicotinoids on mutant C. elegans with increased cuticle permeability An important and poorly defined determinant of potency is the ability of a drug present in the external environment to access the interior of the worm, either by ingestion and absorption across the gut wall or diffusion, and perhaps transport, across the cuticle. In the case of nicotine and neonicotinoids, this may be a limiting factor in terms of their biological activity as they require access to neurotransmitter receptors that are expressed on muscle and neurons. Worm mutants with aberrant cuticular integrity have enhanced sensitivity to drugs32, 33 and provide a useful experimental model to investigate whether drug absorption across the nematode cuticle is a limiting factor in drug sensitivity. The baseline behaviour of the cuticle mutant bus-17 is similar to N2, and it thrashes at a similar rate (Fig. 1b). As before, nicotine and nitenpyram showed a rapid and dose-dependent inhibition of thrashing; however, the potency of both compounds was markedly shifted to lower concentrations (Figs 1b and 2). Remarkably, the bus-17 mutant revealed a susceptibility of C. elegans to thiacloprid and clothianidin that was not apparent in wild-type N2 worms with an intact cuticle (Figs 1b and 2). This increased potency is summarised in concentration–response curves for nicotine, thiacloprid, clothianidin and nitenpyram (Fig. 2). In the bus-17 mutant, thiacloprid is the most potent of the compounds tested, with an EC50 of 480 μm. Figure 2Open in figure viewerPowerPoint Dose–response curves for the effects of nicotinic compounds on motility in a liquid medium of C. elegans. Concentration–response curves for the effects of (a) nicotine, (b) thiacloprid, (c) clothianidin and (d) nitenpyram on thrashing of bus-17 (grey) and wild-type N2 (black) C. elegans. Thrashing rates were generated by taking 60 min (nicotine and nitenpyram) or 120 min (thiacloprid and clothianidin) time points, that is, when the steady state was reached (Fig. 1), and expressed as a percentage of the control thrashing activity. EC50 values (concentration of the drug that produced 50% paralysis) are shown in black for N2 and in grey for bus-17. Data are the mean ± SEM. The EC50 for clothianidin is an approximation, as at the highest concentration tested (2.5 mm) the maximum inhibition observed was 50%. The thrashing assays revealed effects of the compounds on motility over a time course of 4 h, but are not suited for longer drug exposure assays as they are conducted in the absence of the worm's food, E. coli OP50, and therefore over the course of a day control worms will no longer thrash. Therefore, to test whether or not more protracted exposure to the compounds would further increase the sensitivity of C. elegans to the inhibitory effects on motility, i.e. 24 h exposure rather than 4 h exposure, we used an assay in which adult worms were exposed to the drugs on an agar lawn laced with E. coli OP50 (Fig. 3), allowing chronic drug exposure via ingestion and through the cuticle. Again, the potency of nicotine was greater in the bus-17 mutant than in N2, and for clothianidin and thiacloprid revealed a sensitivity that was not evident in N2. An analysis of the concentration–response curves for nicotine, thiacloprid and clothianidin indicates that thiacloprid is the most potent inhibitor of body bends (Fig. 4). Figure 3Open in figure viewerPowerPoint The concentration dependence for the effects of chronic exposure to nicotinic compounds on locomotion of C. elegans. Wild-type (a) and bus-17 (b) worms were exposed for 24 h to varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (control) incorporated into a solid medium. Body bends were counted by visual observation. Data are the mean ± SEM, collected over at least three observations, with the number of replicates given in the graph bars. One-way ANOVA (Kruskal–Wallis test) with Dunn's corrections: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Figure 4Open in figure viewerPowerPoint Concentration–response curves for the effects of chronic nicotine and neonicotinoid compound treatment on locomotion of C. elegans. Concentration–response curves for the effects of (a) nicotine, (b) thiacloprid and (c) clothianidin on body bend rates of bus-17 mutant (grey) and wild-type N2 (black) C. elegans. Body bends rates are expressed as a percentage of the control activity. EC50 values (concentration of the drug that produced 50% paralysis) are shown in black for N2 and in grey for bus-17. Data are the mean ± SEM. The EC50 for clothianidin is an approximation, as at the highest concentration tested (3.75 mm) the maximum inhibition observed was 50%. 3.3 Effects of nicotine and neonicotinoids on C. elegans development During the timeframe of the chronic dosing experiments described above, wild-type N2 worms will typically lay eggs that hatch into larva and then over the course of three further days will mature into adults. We noted during the chronic dosing of adult worms with neonicotinoids that this developmental programme appeared to be delayed by nicotine and thiacloprid, and therefore investigated the impact of these compounds on larval development in more detail. To do this, we monitored the progression of larval development from a synchronised population of eggs through L1, L2, L3 and L4 by counting the relative proportion of each developmental stage after 1, 2, 3 and 6 days exposure to the compounds. Both nicotine and thiacloprid were found to slow larval development (Fig. 5). The differential distribution of distinct larval stages following nicotine and thiacloprid dosing was most marked at day 2, with the effect of thiacloprid the most marked. This suggests that the drug affects L2-to-L3 transition. Clothianidin and nitenpyram at 1 mm failed to shift the development (data not shown). Figure 5Open in figure viewerPowerPoint Effects of nicotine and thiacloprid on the larval development of wild-type C. elegans. Wild-type young hermaphrodites laid eggs on plates dosed with 1 mm of thiacloprid, 1 mm of nicotine or drug vehicle (control). Larval development in the presence of drugs was monitored over time. Worms were assigned to each one of five life stages, namely L1, L2, L3, L4 and gravid adults. The fraction of worms in each stage as a percentage of total population at different time points – 30 h (day 1), 48 h (day 2), 72 h (day 3) and 144 h (day 6) – was measured. Data are shown as the mean of N ≥ 3. 4 DISCUSSION AND CONCLUSIONS To initiate investigations of the poorly understood toxicity of neonicotinoids on important biomass organisms, nematodes, we have exposed the free-living nematode C. elegans to thiacloprid, clothianidin and nitenpyram and to the well-studied nAChR agonist nicotine. Previous studies34 report effects of low mm concentrations of thiacloprid and imidacloprid on reproduction. In our study we investigated effects on motility, and in wild-type N2 worms we observed no significant impairment of locomotion upon acute or chronic exposure to the neonicotinoids thiacloprid or clothianidin, although there was an inhibitory effect of nitenpyram at high mm concentrations. In contrast, nicotine paralysed C. elegans at lower mm concentrations, and it was notable that the potency was 10 times greater with chronic exposure compared with acute assays, consistent with the suggestion that nicotine does not permeate the worm's cuticle readily. To investigate the role of cuticle in drug permeability, we performed locomotion assays on a C. elegans mutant characterised by a 'leakier' cuticle (bus-17).32 We observed clear inhibitory effects of thiacloprid and clothianidin on motility at high micromolar concentrations, with the former being the most potent out of all compounds tested. As the field levels of neonicotinoids in soil and water are orders of magnitude lower (low micromolar concentrations in water and 2 ng g−1 in soil),35 at least short-term exposure to neonicotinoids should have a limited effect on related nematodes, assuming that N2 C. elegans is a predictor for wild isolates and nematode species. However, protracted incubation with nicotine and thiacloprid delayed larval development of wild-type N2, suggesting that compounds may accumulate in the nematode's tissues, highlighting the importance of bioaccumulation in determination of ecotoxicity. Moreover, neonicotinoids have been shown to disrupt aspects of movement in bees36 and in C. elegans37 (this study). In bumblebees there is also evidence for disruption of higher cognitive tasks by neonicotinoids,28 and therefore the question as to whether neonicotinoids affect the more intricate behavioural repertoires in C. elegans is also worthy of further investigation. Overall, our data highlight that C. elegans is not susceptible to field-relevant concentrations of neonicotinoids, despite the fact that susceptibility to high concentrations of all three of the neonicotinoids tested can be demonstrated in a mutant with increased cuticle permeability. Indeed, our data suggest that neonicotinoids are three orders of magnitude less potent on C. elegans than on insects.27-29, 38-46 This creates an opportunity to use a C. elegans transgenic model hopping approach that utilises the introduction of genes of interest, from either pest species or other non-target organisms, into wild-type or mutant C. elegans to provide an experimentally tractable system to investigate mode of action and selective toxicity.47-49 This approach will use manipulation of the insect genes encoding candidate neonicotinoid-sensitive nAChRs. As C. elegans possess essential biosynthetic machinery for the invertebrate nAChR receptor,23, 24 this creates a favourable cellular environment for receptor expression. Potential candidate insect nAChR subunits are brown planthopper Nilaparvata lugens α150 and green peach aphid Myzus persicae β1, subunits both associated with neonicotinoid resistance, as well as housefly Musca domestica α1, linked to neonicotinoid susceptibility.51 The data shown in this study indicate that the C. elegans bus-17 mutant will provide a useful genetic background for these studies by enhancing the access of neonicotinoid compounds to their target receptor expressed in the worm. Therefore, by expressing individual insect subunits in the bus-17 background in specific tissues in C. elegans, we can investigate the neonicotinoid sensitivity of the insect receptor subunits47 by comparing the neonicotinoid sensitivity of transgenic versus control strains in behavioural assays. Such pharmacological characterisation is crucial in understanding neonicotinoid-induced behavioural alterations and may contribute to the identification of new, more selective neonicotinoids. ACKNOWLEDGEMENTS This research was financially supported by the Gerald Kerkut Charitable Trust and the University of Southampton. Strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programmes (P40 OD010440). The authors would like to thank Dr Anna Crisford and Dr Fernando Calahorro for continual guidance and assistance. REFERENCES 1Jeschke P, Nauen R, Schindler M and Elbert A, Overview of the status and global strategy for neonicotinoids. J Agric Food Chem 59: 2897– 2908 (2011). 2Elbert A, Haas M, Springer B, Thielert W and Nauen R, Applied aspects of neonicotinoid uses in crop protection. Pest Manag Sci 64: 1099– 1105 (2008). 3Tomizawa M and Casida JE, Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. 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