03-results-01.Rmd 77.2 KB
 mk11g11 committed Sep 16, 2019 1 # Effects of neonicotinoids on the behaviour and development of *C. elegans* {#results-1}  ab604 committed Aug 17, 2019 2   mk11g11 committed Sep 16, 2019 3 4 5 6 7 8 9 10 11 12 13 14 15 {r echo=FALSE, include=FALSE} library(cowplot) library(tidyverse) library(ggpubr) library(readr) library(ggplot2) library(scales) library(curl) library(devtools) library(knitr)  ## Introduction  mk11g11 committed Oct 06, 2019 16 Neonicotinoids are the most commonly used insecticides worldwide due to their high efficacy against pest insects (Section \@ref(potentpests)), selective toxicity to insect pests over mammals (Section \@ref(seltox)) and advantageous physicochemical attributes (Section \@ref(physchem)). The main disadvantage of these compounds is that they can be toxic to non-target species, including bees (Section \@ref(sublethal)). This undesired ecotoxicological effect spurred a debate over their environmental impact and revealed a necessity to further investigate their effects on other ecologically important organisms such as worms.  mk11g11 committed Sep 16, 2019 17   mk11g11 committed Apr 13, 2020 18 ## Ecological role of non-parasitic worms ##{#ecologoicalroleofwormschapter3}  mk11g11 committed Sep 29, 2019 19   mk11g11 committed Feb 19, 2020 20 Non parasitic earth worms and nematodes, play an important biological role. They are the most abundant multicellular organisms on earth and are significant biomass contributors. In addition, they cycle nutrients contributing as much as 1/5 of all bioavailable nitrogen in soil [@neher2001], promoting plant growth [@ingham1985] and soil fertility. They are also valuable bioindicators and have been used in the assessment of contaminated soil [@lecomte-pradines2014].  mk11g11 committed Sep 16, 2019 21   mk11g11 committed Feb 19, 2020 22 ## Residues of neonicotinoids in soil  mk11g11 committed Apr 13, 2020 23 Neonicotinoids are commonly applied as seed dressing [@jeschke2011; @alford2017], due to a benefit of extended crops protection resulting in a reduction in the insecticide application frequency. However, on average, only 5 % of the active ingredient is taken up by and distributed throughout the developing plant [@sur2003]. The remainder enters the wider environment, including soils, where they can have a negative effect on inhabiting worm species.  mk11g11 committed Sep 16, 2019 24   mk11g11 committed Apr 13, 2020 25 The levels of neonicotinoids in terrestrial terrains vary depending on the composition and the physical properties of the soil [@moertl2016; @selim2010; @zhang2018]. Numerous studies investigated their levels in various soil types, following variable post planting period and generally report the sub $\mu$M concentrations (reviewed in @wood2017). However, they persist in terrestrial terrains from a few days to several years (reviewed in @goulson2013). Nitenpyram and thiacloprid typically remain there for several weeks, clothianidin for just over a year, whereas imidacloprid for several years. Long dissipation half-life and absorption capacity means that neonicotinoids may come in contact with soil- residing worms for prolonged time periods. Neonicotinoids can also enter the worm’s interior by multiple routes. They may diffuse across the worm’s cuticle, or be ingested with soil particles [@pisa2015]. Exposure to residual neonicotinoids can have a negative impact on many aspects of worm’s biology.  mk11g11 committed Sep 16, 2019 26 27 28 29 30 31   mk11g11 committed Feb 19, 2020 32 33 ## Cholinergic regulation of worm behaviour ###{#cholinericwormbeh} The cholinergic system is the primary target of neonicotinoids in insects (Section \@ref(neonicstarget)). Acetylcholine is pivotal in regulation of worm behaviour (Section \@ref(cholinergicneurotransmissioninworms)). Most of the current knowledge is derived from work on the soil nematode and model organism *C.elegans*.  mk11g11 committed Sep 16, 2019 34 35  #### Locomotion ####{#locomotion}  mk11g11 committed Feb 19, 2020 36 37 *C. elegans* exhibits distinct locomotory behaviours in liquid and on solid medium (Section \@ref(locomotionbehaviour)). In the liquid medium, it flexes back and forth, whereas on solid medium it crawls is a sinusoidal fashion. These behaviours are mediated by the body musculature composed of 95 muscle cells. The muscle cells are arranged into 4 muscle rows: a pair of longitudinal ventral rows and a pair of dorsal rows. Their function is under the control of the nervous system (Figure \@ref(fig:motility-intro-label)). There are 4 motor neuron classes synapsing onto the dorsal muscle (AS, DA, DB, and DD) and 4 innervating the ventral muscle (VA, VB, VC, and VD). Motor neurons belonging to class A, B and AS release ACh and are excitatory, whereas motor neurons of class D are inhibitory and release GABA [@mcintire1993]. Bending of the dorsal side is associated with excitation and contraction of the dorsal side and simultaneous inhibition and relaxation of the ventral side, whereas the reverse is true when the ventral side bends. A and B neurons not only innervate muscle, but also send out processes to the collateral side, and synapse onto D, inhibitory neurons [@white1986]. By doing so, acetylcholine acts directly on the muscle to elicit contraction and indirectly to relax the opposite side. Taken together, this allows the bending of a particular portion of one and the relaxation of the opposing side of the body to enable the worm’s locomotory activities. Whereas the propagation of the electrical signal down the axis of the muscle whilst on solid medium, results in forward movement.  mk11g11 committed Sep 16, 2019 38   mk11g11 committed Feb 19, 2020 39 40   mk11g11 committed Sep 16, 2019 41 42  ##### Regulation of the direction of movement  mk11g11 committed Feb 19, 2020 43 The direction of worm’s movement is controlled by so called command interneurons [@chalfie1985; @white1986]. There are 5 command interneurons, namely PVC, AVB, AVA, AVD and AVE. These make synaptic connections with appropriate motor neurons of the BWM. PVC and AVB innervate VB and DB neurons which regulate forward movement. AVA, AVD and AVE innervate VA and VB which regulate backward movement [@chalfie1985; @white1986].  mk11g11 committed Sep 16, 2019 44   mk11g11 committed Oct 06, 2019 45 #### Sensory regulation of the locomotion ####{#sensoryregulation}  mk11g11 committed Feb 19, 2020 46 Locomotion can be regulated by the environmental cues detected by the sensory neurons which relay information into the locomotory circuitry. Locomotion on solid medium is greatly influenced by the present of food [@dalliere2017]. Whilst on food, *C. elegans* exhibits two types of locomotory behaviour: dwelling and roaming. Dwelling is characterised by enhanced turning frequency but low movement speed rate, whereas roaming is associated with decreased turning frequency but higher movement speed. Upon transfer to the area with no food source, worms search for food evidented by enhanced movement speed. *C. elegans* locomotion is also influenced by noxious stimuli and olfactory cues. For example, in response to a range of nociceptive stimuli ASH head neurons are activated [@hilliard2005]. This leads to rapid and transient backward movement, followed by a change of direction in the forward movement (reviewed in @bono2005).  mk11g11 committed Sep 16, 2019 47 48 49 50 51 52 53  (ref:motility-circuit) **Locomotory circuit in *C. elegans*.** Release of acetylcholine onto dorsal muscles (+) leads to their excitation and contraction. At the same time, acetylcholine activates GABAergic neurons contralaterally. Release of GABA leads to inhibition of ventral muscles. The signal propagates down the axis and leads to coordinated sinusoidal movement. Figure taken from www.wormatlas.org. {r motility-intro-label, fig.cap= "(ref:motility-circuit)", fig.scap= "Locomotory circuit in \\textit{C. elegans}.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/intro_2/motility.jpg")   mk11g11 committed Feb 19, 2020 54 55 \newpage  mk11g11 committed Sep 16, 2019 56 #### Egg laying  mk11g11 committed Apr 13, 2020 57 Egg-laying is controlled by the contraction of vulvar muscles under the influence of the nervous system (Figure \@ref(fig:egg-laying-label)). The main excitatory neurotransmitter is 5-HT [@waggoner1998] released from the Hermaphrodite Specific Neurons (HSNs). There are other neurotransmitters involved, such as excitatory acetylcholine [@trent1983] released from the Ventral C neurons (VCs). In addition, there are four uv1 neuroendicrine cells linking uterus and vulva which release tyramine to inhibit egg laying [@alkema2005].  mk11g11 committed Sep 16, 2019 58 59 60 61  (ref:Egg-laying-fig) **Neuronal circuitry of *C. elegans* vulva.** Lateral image of the *C. elegans* hermaphrodite (top) and positioning of the vulva (bottom). 16 vulval muscles, out of which vm1 and vm2 are the most important, contract to expel eggs out. HNS and VC neurons synapse onto vm2 muscle. Uv1 neuroendocrine cells link the uterus and vulva and inhibit egg-laying. Image taken from [@collins2016]. {r egg-laying-label, fig.cap="(ref:Egg-laying-fig)", fig.scap= "Neuronal circuitry of \\textit{C. elegans} vulva.", echo=FALSE, out.width='100%', fig.align='center',}  mk11g11 committed Feb 19, 2020 62 knitr::include_graphics("fig/intro_2/vulva.png")  mk11g11 committed Sep 16, 2019 63 64 65 66 67 68 69 70 71 72   mk11g11 committed Apr 13, 2020 73 74 75 76 ### Effects of neonicotinoids on worms ###{#chapter3effectsofneonics} There is a limited literature regarding the effects of neonicotinoids on earthworms and nematodesn (Section \@ref(effectsofneonicsonworms)). In earth worms negative effects of neonicotinoid on the reproduction [@gomez-eyles2009; @baylay2012; @kreutzweiser2008; @alves2013], avoidance [@alves2013] and locomotion [@dittbrenner2011] have been described. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of these insecticides. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014], whereas thiaclopand inhibits its egg-hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. They seem to have variable effects on *C. elegans*. @mugova2018 reports an inhibitory effect on motility of imidacloprid at concentration ranging from 120 $\mu$M to 2 mM. Thiacloprid seems to have an opposite effect. At concentrations ranging from 2 to 40 $\mu$M it elevates locomotion in liquid of mixed developmental stage population of *C. elegans* [@hopewell2017]. Variable effects of neonicotinoids on egg-laying are also reported. Low mM concentrations of clothianidin and thiacloprid inhibit egg-laying [@gomez-amaro2015]. In contrast, imidacloprid at a single concentration of 20 nM, elevates the number of egg-laid, but has no effect at 120 $\mu$M - 2 mM, suggesting this effect is not dose-dependent [@ruan2009].  mk11g11 committed Sep 16, 2019 77 78 79 80 81 82 83 ### Chapter aims The aim of this chapter is to better understand the effects of neonicotinoids on Nematoda representative *C. elegans*. Their impact on defined and well understood behaviors governed by the cholinergic transmission are tested and compared to the effects elicited by a classical nAChR agonist nicotine. This will be used to inform the potential risk of these environmental neurotoxins against Nematoda and on their mode of action. \newpage ## Results  mk11g11 committed Feb 19, 2020 84 ### Effects of nicotine on thrashing  mk11g11 committed Oct 06, 2019 85 Upon immersion of *C. elegans* in liquid, it exhibits rhythmic swimming-like locomotory behaviour driven by the body wall musculature and inputs from the central nervous system (Section \@ref(locomotion)). This locomotory behavior is known as thrashing. It can be easily scored and used as an assessment for the effects of acute exposure to nicotine and neonicotinoid on motility of *C. elegans*. Worms placed in untreated liquid medium retain a constant thrashing rate of ~40-45 repeats per 30s throughout the duration of the experiment (2 hours) (Figure \@ref(fig:thrashing-nicotine)). Addition of nicotine at concentrations ranging from 1 to 100 mM leads to dose-dependent inhibition of motility (Figure \@ref(fig:thrashing-nicotine)). The time course for the effects of nicotine varied dependent on the concentration. At doses eliciting partial paralysis (i.e. 10 and 25 mM) 2 “phases” of inhibition can be seen. The immediate one seen after 10 minutes which recovers slightly, and the second which reaches a steady state inhibition after 60 minutes. Nicotine at 100 mM led to a complete inhibition of thrashing. This effect was visible 10-minutes post exposure and sustained for 2 hours.  mk11g11 committed Sep 16, 2019 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 The EC~50~ of nicotine on thrashing was 26.2 (95% CI= 17.4 to 38.8) mM, respectively) (Figure \@ref(fig:thrashing-nicotine)b). This low efficacy may be due to reduced bioavailability of nicotine in the worm. (ref:thrashing-data) **Concentration and time dependence of the effects of nicotine on thrashing of *C. elegans*.** a) Wild type N2 worms were exposed to varying concentrations of nicotine. The number of thrashes were recorded for 30 seconds at the indicated time points. b) Concentration dependence of nicotine inhibition of thrashing on wild-type N2 worms. Dose-response curve were generated by taking the 60 minute time- points; that is when the steady-state inhibition of thrashing was reached, and expressed as % of control thrashing. EC~50~ values (dose at which thrashing was reduced by half) is shown. Data are mean $\pm$ SEM of $\ge$ 14 individual worms collected from experiments done on 3 days. {r thrashing-nicotine, fig.cap= "(ref:thrashing-data)", fig.scap = "Concentration and time dependence of the effects of nicotine on thrashing of \\textit{C. elegans}.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results2/final/pngpdf/thrashing_nic_1.png")  \newpage #### Effects of pH on nicotine induced inhibition of thrashing Bioavailability of compounds might be impaired by the physicochemical properties of drugs, such as charge. Nicotine is a diprotic base, with pKa of pyridine ring of 3.12 and pKa of pyrrolidine ring of 8.02 [@ciolino1999]. By altering the pH of liquid medium from 7 to 6 and 9, the equilibrium between charged and uncharged nicotine species shifts. One might predict this has an effect on the efficacy of nicotine. Indeed, The EC~50~ of nicotine at pH= 6 and 9 is slightly, but not significantly decreased in comparison to pH=7 (16.7 (95% CI= 11.6 to 23.6), 15.2 (95% CI= 11.0 to 20.5) and 26.2 (95% CI= 17.4 to 38.8) mM, respectively). Since pH of the external buffer does not have a marked effect on efficacy, all following experiments were performed at neutral pH. (ref:nicotine-ph) **Effects of pH on the concentration dependence for the effects of nicotine on *C. elegans* thrashing.** Dose-response curves for the effects of pH on efficacy of nicotine on thrashing. Wild-type worms ere exposed to varying concentrations of nicotine suspended in a buffer at pH = 7, 6 and 9. The number of thrashes were scored for 30 seconds at 60 minute time-point. Data is expressed as % of control thrashing. EC~50~ values are shown in black for N2 pH=7, red for N2 pH=6 and purple for N2 pH=9. Data are mean $\pm$ SEM of $\ge$ 11 individual worms collected from paired experiments done on 3 separate days. {r thraahing-ph-label, fig.cap="(ref:nicotine-ph)", fig.scap= "Effects of pH on the concentration dependence for the effects of nicotine on \\textit{C. elegans} thrashing.", fig.align='center', out.width='70%', echo=FALSE}  mk11g11 committed Feb 19, 2020 102 knitr::include_graphics("fig/results2/final/pngpdf/Nic-DR-thrashing-all-n2.png")  mk11g11 committed Sep 16, 2019 103 104 105 106 107  \newpage ### Effects of the cuticle on nicotine induced inhibition of thrashing  mk11g11 committed Feb 19, 2020 108 The second factor limiting drugs’ bioavailability is worm’s cuticle. This idea is supported by the observation that the application of 0.1mM of nicotine on intact worm has no effects on thrashing (Figure \@ref(fig:thrashing-nicotine)), but when applied on the isolated body wall muscle or dissected worm, it induced large inward current and paralysis, respectively [@richmond1999; @matta2007]. This suggests that the cuticle is a major physical barrier for drug entry. To provide a platform for investigation of the importance of the cuticle in drug permeability, *C. elegans* *bus-17* mutant strain was used. *Bus-17* is a GT13 glycosyltransferase mutant [@yook2007] exhibiting defective glycoprotein production resulting in abnormal surface coat and altered cuticular integrity.  mk11g11 committed Sep 16, 2019 109 110 111 112 Mutation of *bus-17* genes have no significant effect on the thrashing behavior of *C. elegans* frequency. Worms retained a constant thrashing rate of 40-45 thrashes per 30s over the duration of the experiment (Figure \@ref(fig:thrashing-cuticle-label)). Immersion in nicotine led to concentration dependent paralysis, but the potency of nicotine on the mutant vs the wild-type strain is almost 10-fold greater (Figure \@ref(fig:thrashing-cuticle-label)). Moreover, the inhibitory effects of nicotine at 10 and 25 mM on *bus-17* worms lacks 2 phases of inhibition seen previously. This might suggest that nicotine reaches the internal molecular targets more quickly. (ref:thrashing-cuticle) **The effects of the cuticle on the concentration and time dependence of nicotine inhibition of *C. elegans* thrashing.** N2 wild type (a) and *bus-17* mutant (b) worms were exposed to varying concentrations of nicotine. The number of thrashes were recorded for 30 seconds at the indicated time points. b) Concentration dependence of nicotine dependent inhibition of thrashing on N2 (black) and *bus-17* worms (grey). Dose-response curve were generated by taking the 120-minute time- points; that is when the steady-state of thrashing inhibition was reached, and expressed as % of control thrashing. EC~50~ values are shown on graphs. Data are mean $\pm$ SEM of $\ge$ 15 individual worms collected from paired experiments done on 3 days.  mk11g11 committed Apr 13, 2020 113 {r thrashing-cuticle-label, fig.cap= "(ref:thrashing-cuticle)", fig.scap = " The effects of the cuticle on the concentration and time dependence of nicotine inhibition of \\textit{C. elegans} thrashing.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 16, 2019 114 115 116 117 118 knitr::include_graphics("fig/results2/final/pngpdf/fig2.png")  \newpage  mk11g11 committed Sep 29, 2019 119 ### Effects of neonicotinoids on thrashing ###{#effectsofneonicsonthrashing}  mk11g11 committed Sep 16, 2019 120 To assess the effects of neonicotinoids on motility of worms in liquid, the thrashing experiment was repeated with nitenpyram, thiacloprid and clothianidin. Out of the three compounds tested, only nitenpyram at concentrations ranging from 1 to 100 mM induced concentration-dependent paralysis of N2 wild-type worms. Low water solubility of thiacloprid and clothianidin limited the maximum testable doses to 1.5 and 2.5 mM, respectively. Results in (Figure \@ref(fig:thrashing-tc-comp-label), left panel), show that at these doses neither of the two have an effect on thrashing of wild-type worm.  mk11g11 committed Sep 29, 2019 121 To determine whether a cuticle also limits the bioavailability of neonicotinoids, experiments were repeated on *bus-17* mutant. Shift in potency of all compounds was noted (Figure \@ref(fig:thrashing-tc-comp-label) and \@ref(fig:DR-neonics-label)). The EC~50~ of nitenpyram on wild-type increased by almost 12-fold on mutant worm (195.8 (95% CI= 133.9 to 313.9) and 16.6 (95% CI= 12.0 to 22.6) mM). Thiacloprid and clothianidin were with no effects on wild-type worms, but induced paralysis of the *bus-17* mutant with the EC~50~ of 377.6 $\mu$M (95% CI= 311.8 to 454.0 $\mu$M) and 3.5 mM (95% CI= 24.1 to 53.5mM), respectively. The time course for both clothianidin and thiacloprid have similar features: gradual increase in inhibition of thrashing with the maximal effect achieved after 1 hour followed by a slow retrieval. The gradual recovery might represent adaptation of the neuronal circuit for locomotion, desensitization of receptors mediating the response or precipitation of a drug (although no visual sigh of this were observed with exception of 1.5 mM thiacloprid after 45 minutes). The breakdown in liquid is unlikely, as both compounds have long half-live in water (Table \@ref(tab:properties)) [@gilbert2010].  mk11g11 committed Sep 16, 2019 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137  (ref:thrashing-tc-comp-capt) **The concentration and time dependence of neonicotinoids inhibition of *C. elegans* thrashing.** Wild type (left panel) and *bus-17* (right panel) worms were acutely exposed to varying concentrations of nitenpyram, thiacloprid, clothianidin or drug vehicle (0, Ctr). The number of thrashed over 30 seconds at indicated time points was scored. Data are mean $\pm$ SEM of $\ge$ 6 individual worms collected from paired experiments done on $\ge$ 2 days. {r thrashing-tc-comp-label, fig.cap= "(ref:thrashing-tc-comp-capt)", fig.scap= "The concentration and time dependence of neonicotinoids inhibition of \\textit{C. elegans} thrashing.", fig.width=10, fig.asp=1.1, fig.align='center', echo=FALSE} knitr::include_graphics("fig/results2/final/pngpdf/Fig3.png")  (ref:DR-neonics) **Dose-response curves for the effects of neonicotinoids on *C. elegans* thrashing.** Concentration-response curves for the effects of nitenpyram (a), thiacloprid (b) and clothianidin (c) on thrashing of wild-type (black) and *bus-17* (grey) *C. elegans*. Dose-response curves were generated by taking 120-minute time-point for nitenpyram and 120-minute time points for thiacloprid and clothianidin; that is when the steady-state inhibition of thrashing was reached, and expressed as % control thrashing. Data and mean $\pm$ SEM. The EC~50~ for clothianidin are approximations, because the highest concentration tested (2.5 mM) inhibited thrashing by 55 % in *bus-17*. {r DR-neonics-label, fig.cap="(ref:DR-neonics)", fig.scap = "Dose-response curves for the effects of neonicotinoids on \\textit{C. elegans} thrashing.", fig.width=10, fig.asp=1.1, fig.align='center', echo=FALSE} knitr::include_graphics("fig/results2/final/pngpdf/Fig4.png")  \newpage ### Kinetic properties of nicotine- and neonicotinoid- induced inhibition of thrashing  mk11g11 committed Feb 19, 2020 138 To observe penetration properties of compounds, the thrashing experiment was repeated in the presence of drug doses inducing inhibition of thrashing. The observations at early time points were made to determine how quickly a maximum effect can be observed (Figure \@ref(fig:onset-plot-label)). High doses of nicotine paralysed all N2 wild-type and *bus-17* mutant worms within 6 minutes and time taken to paralyse 50 % of worms (t~1/2~) of less than a minute. Since neonicotinoids did not induce full paralysis of wild-type worms, only the effects of thiacloprid and nitenpyram on *bus-17* mutant were investigated. The action of neonicotinoids was much slower when compared to nicotine. Worms became immobile after 1 hour of incubation, and the t~1/2~ for both compounds was extended to 5 minutes.  mk11g11 committed Sep 16, 2019 139 140 141 142 143 144 145 146 147  (ref:onset-plot-capt) **The onset kinetics of nicotine and neonicotinoid induced inhibition of *C. elegans* thrashing.** Worms were submerged in drug concentrations at which complete paralysis was achieved; that is: a) N2 wild-type in 100mM nicotine, b) *bus-17* mutant in 25mM nicotine, c) *bus-17* mutant in 50 mM nitenpyram and d) *bus-17* mutant in 1mM thiacloprid. The number of thrashes over 30 seconds at the indicated time points were counted. Data are mean $\pm$ SEM of $\ge$ 4 individual worms collected from pared experiments done on 2 days. Note a different time scale in a, b compared to c and d. {r onset-plot-label, fig.cap= "(ref:onset-plot-capt)", fig.scap = "The onset kinetics of nicotine and neonicotinoid induced inhibition of \\textit{C. elegans} thrashing.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results2/final/pngpdf/onset.png")  \newpage  mk11g11 committed Feb 19, 2020 148 Recovery assay gives an indication of how quickly the effects of compounds reverse. This reversal when drug inhibited worms are moved into a larger volume of drug free are thought to be due to a diffusion out of the worm or/and drug metabolism via various detoxifying systems [@lindblom2006]. In this experiment, worms were placed in drug concentration that induced full paralysis for 20 minutes in the case of nicotine and 60 minutes in case of nitenpyram and thiacloprid. Once paralysis was achieved, worms were transferred to drug-free medium and the thrashing rates were monitored over time (Figure \@ref(fig:merged-recovery-title)). Following, the exposure to high concentration of nicotine and 2.5 hours of washing, a proportion of worms remained paralysed: 50% of wild-type and 25% of *bus-17* (data not shown). The remaining wild-type and *bus-17* worms recovered with t~1/2~ of 1.5 hours and 50 minutes respectively.  mk11g11 committed Sep 16, 2019 149 150 In contrast, all worms paralysed by nitenpyram or thiacloprid returned to normal basal thrashing within 2 hours of washing. The time taken for half recovery for both compounds was 1 hour.  mk11g11 committed Feb 19, 2020 151 (ref:recovery-thrashing) **Recovery kinetics of nicotine and neonicotinoid-paralysed *C. elegans*.** N2 wild-type and *bus-17* mutant worms were exposed to indicated concentrations of nicotine (a and b), nitenpyram (c) and thiacloprid (d). Paralysed worms were transformed to drug-free dish and recovery was scored by noting a number of thrashes / 30s. Only worms recovered are included in this analysis. Alongside these, worms were transferred from drug-containing to drug containing dish (+ve ctr ) and from drug-free to drug-free dish (-ve ctr). Data are mean $\pm$ SEM of 8 individual worms collected from experiments done $\ge$ 2 days.  mk11g11 committed Sep 16, 2019 152   mk11g11 committed Apr 13, 2020 153 {r merged-recovery-title, fig.cap= "(ref:recovery-thrashing)", fig.scap= "Recovery kinetics of nicotine and nonicotinoid-paralysed \\textit{C. elegans}.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 16, 2019 154 155 156 157 158 159  knitr::include_graphics("fig/results2/final/pngpdf/recovery.png")  \newpage  mk11g11 committed Feb 19, 2020 160 161 162 163 164 165 166 167 168 169 170 171 172 173 #### Effects of nicotine and neonicotinoids on *C. elegans* lenght During acute and chronic exposure experiments, it was noted that a 4 hour incubation of worms with high nicotine concentrations had marked effect on morphology. Specifically, nicotine at concentrations inducing paralysis led to shrinking of the worm. To investigate this further and determine if neonicotinoids have the same effect, worms were exposed to either drug concentration inducing paralysis, or the highest possible testable concentrations. To maximise the concentration of drugs inside the worm, *bus-17* mutant was used in this experiment. The images of L4 + 1 incubated with nicotine or neonicotinoids for 4 hours were taken and the measurements of the length of the body were made. Exposure to 25 mM nicotine led to reduction in the body lenght. In contrast, neonicotinoids had no effect (Figure \@ref(fig:shrinking-title1)). \newpage (ref:shrinking) **Effects of nicotinic compounds on *C. elegans* body length.** *Bus-17* mutant was submerged for 4 hours in solution containing 25 mM nicotine, 50 mM nitenpyram, 1.5 mM thiacloprid or 2.5 mM clothianidin or vehicle control (Ctr). 4 hours later, the images of worms were taken and the body length measured. Data are mean $\pm$ SEM. Number of determinations $\ge$ 12 collected over 3 observations. One way ANOVA with Bonferonni corrections, $**$ P $\le$ 0.01.  mk11g11 committed Apr 13, 2020 174 {r shrinking-title1, fig.cap="(ref:shrinking)", fig.scap = " Effects of nicotinic compounds on \\textit{C. elegans} body lenght.", fig.align='center', echo=FALSE,}  mk11g11 committed Feb 19, 2020 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209  # cbp1 <- c("#999999", "#E69F00", "#56B4E9", "#009E73", # "#F0E442", "#0072B2", "#D55E00", "#CC79A7") # # data19 <- read_csv("Analysis/Data/Transformed/Shrinking.csv") # shrinking_tidy <- data19 %>% # drop_na() %>% # mutate (Conc = factor(Concentration, # levels = c("Ctr", "Nitcotine_25mM", "Nitenpyram_50mM", "Thiacloprid_1.5mM", "Clothianidin_2.5mM"), # labels = c("Ctr", "Nicotine 25 mM", "Nitenpyram 50 mM", "Thiacloprid 1.5 mM", "Clothianidin 2.5 mM"))) # # shrinking_stats <- shrinking_tidy %>% # group_by(Conc) %>% # summarise(mean_length=mean(length), # sd=sd(length), # se=sd/sqrt(length(length))) # # shrinking_plot <- shrinking_stats %>% # ggplot(aes(x = Conc, # y = mean_length, fill=Conc)) + # scale_fill_manual(values = cbp1) + # geom_bar(stat = "identity", colour="black") + # geom_errorbar(aes(ymin = mean_length-se, ymax = mean_length+se), width=0.4) + # ylab("Body length (mm)") + # labs(fill = "") + # ylim(0, 1.5) + # annotate("text", x=2, y=1.5, label= "**") + # theme(axis.text.x = element_blank(), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # axis.ticks.x = element_blank(), # axis.title.x = element_blank()) + # ggsave("fig/results2/bodylengthploy.pdf") knitr::include_graphics("fig/results2/final/pngpdf/bodylengthploy.png")  mk11g11 committed Sep 16, 2019 210 211 212 213 214   \newpage  mk11g11 committed Feb 19, 2020 215 216 ### Effects of chronic exposure of *C. elegans* to nicotine and neonicotinoids on behaviour Liquid assays allow for relatively short-term exposure. To test whether protracted exposure of worms provides a better paradigm for sensitivity on-plate assay was employed (Section \@ref(onplateassay)). The concentrations used were ranged 0.5 to 25 mM nicotine, 1 mM nitenpyram, 1 $\mu$M to 1.5 mM thiacloprid and 0.5 to 3.75 mM clothianidin. Worms were exposed to treatment for a period of 24-hours and their effects on locomotion and fertility were measured. This allows for prolonged drug exposure which may lead to accumulation of the drug inside the worm and increased efficacy of compounds on worm behavior.  mk11g11 committed Sep 16, 2019 217 218 219 220  #### Effects of nicotine on avoidance During the experimentation, an observation that the proportion of worms disappeared from nicotine containing plates was made. After 24 hour incubation with nicotine at concentrations $\ge$ 25 mM, the number of worms remaining on the plate was significantly reduced in comparison to the control (Figure \@ref(fig:avoid-label). Closer observation revealed that in the presence of nicotine, worms escaped the experimental arena by crawling to the side of the plate. Neither of the three neonicotinoids had such effect (data not shown).  mk11g11 committed Feb 19, 2020 221 222 223 224 225 226 227 228 229 230 231 232  \newpage  mk11g11 committed Sep 16, 2019 233   mk11g11 committed Feb 19, 2020 234 235 (ref:avoid) **The concentration dependence of the effects of nicotine on *C. elegans* avoidance.** 4-10 wild-type worms were placed on agar plate containing indicated nicotine concentrations or drug vehicle (0). 24 hours later, the % of worms remaining on the plate was scored. Data are mean $\pm$ SEM, collected from 2 - 4 individual experiments. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.  mk11g11 committed Apr 13, 2020 236 {r avoid-label, fig.cap="(ref:avoid)", fig.scap= " The concentration dependence of the effects of nicotine on \\textit{C. elegans} avoidance.", fig.align='center', echo=FALSE}  mk11g11 committed Feb 19, 2020 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269  # ann_text_avoid_1 <- data.frame(Dose = factor(c(25, 50, 100), levels = c(25, 50, 100)), # mean_readout=100, # lab_avoid_1 = c("****", "***", "****"), # Exp = as.factor(42)) # # # avoid <- on_plate_dat_trans_1 %>% # filter(Experiment == 41) %>% # group_by(Dose) %>% # summarise(mean_readout= mean(readout), # n = n(), # sd=sd(readout), # se=sd/sqrt(length(readout))) %>% # ggplot (aes(x = Dose, y = mean_readout, fill = Dose)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # scale_fill_manual(values=c('#000000','#333333', '#666666','#999999', '#CCCCCC', '#D3D3D3', '#DCDCDC')) + # ylim(0, 100) + # geom_text(data = ann_text_avoid_1, aes(label = lab_avoid_1)) + # ylab("% worms on plate") + # xlab("Nicotine, [mM]") + # theme(axis.text = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # axis.title = element_text(size=12), # text = element_text(size=12, family="sans")) + # ggsave("fig/results2/final/pngpdf/avoidancegraph.pdf") knitr::include_graphics("fig/results2/final/pngpdf/avoidancegraph.png")  mk11g11 committed Sep 16, 2019 270 271 272 273  \newpage  mk11g11 committed Sep 29, 2019 274 #### Effects on body bends ####{#bodybendsneonics}  mk11g11 committed Oct 06, 2019 275 Whilst on solid medium, *C. elegans* exhibits sinusoidal movement (Section \@ref(locomotionbehaviour)). This can be quantified by counting a number of forward body bends per unit of time and is a measure of the motor function. The presence of food modifies this behavior (Section \@ref(sensoryregulation)), therefore the measurements were made on treatment - soaked solid medium containing no OP50 food patch (Section \@ref(onplateassay)).  mk11g11 committed Sep 16, 2019 276 277 278  Untreated wild-type worms move at a rate of 39 body bends per minute (Figure \@ref(fig:BB-plot-label), left panel). This is reduced to 33 bends per minute in *bus-17* mutant (Figure \@ref(fig:BB-plot-label) right panel), due to a reduced traction of the body on agar medium [@yook2007]. The body bends of wild-type *C. elegans* was altered by nicotine with with the EC~50~ of 3.6 mM (95 % CI= 2.6 to 4.4 mM), whereas nitenpyram, thiacloprid and clothianidin had no effect (Figure \@ref(fig:BB-plot-label) and \@ref(fig:DR-body-bends-label)). In contrast, the body bends rate of *bus-17* mutant was reduced by all compounds, except for up to the 1 mM nitenpyram. The EC~50~ for the effects of nicotine and clothianidin was 1.6 (CI= ) and 3.3 (CI = ) mM, respectively. Thiacloprid was the most potent with the EC~50~ of 721.2 $\mu$M (95 % CL= 502.4 μM to 1.0 mM) (Figure \@ref(fig:DR-body-bends-label)).  mk11g11 committed Feb 19, 2020 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304   mk11g11 committed Apr 13, 2020 305 (ref:BB-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoid on body bends of *C. elegans*.** N2 wild-type (left panel) and *bus-17* mutant (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, nitenpyram, thiacloprid, clothianidin or drug vehicle (O), incorporated into solid medium. Body bends were counted by visual observation. Data are mean $\pm$ SEM of $\ge$ 5 individual worms collected from $\ge$ 3 paired experiments. One way ANOVA (Kruskal-Wallis test) with Dunn’s Corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.  mk11g11 committed Feb 19, 2020 306   mk11g11 committed Apr 13, 2020 307 {r BB-plot-label, fig.cap= "(ref:BB-plot-capt)", fig.scap= " The concentration dependence for the effects of nicotine and neonicotinoid on body bends of \\textit{C. elegans}.", fig.align='center', include=TRUE, echo=FALSE}  mk11g11 committed Sep 16, 2019 308 309 #Plot body bends graphs #make a data frame for facet labels  mk11g11 committed Feb 19, 2020 310 # labelsBB <- c("9" = "Nicotine N2", "10" = "Nicotine bus17", "11"= "Nitenpyram N2", "12" = "Nitenpyram bus17", "13" = "Thiacloprid N2", "14" = "Thiacloprid bus17", "15" = "Clothianidin N2", "16" = "Clothianidin bus17")  mk11g11 committed Sep 16, 2019 311 #labels for sagnificance to be ploted on a graph  mk11g11 committed Feb 19, 2020 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 # ann_text <- # data.frame( # Dose = factor (c(1, 10, 25), levels = c("1", "10", "25")), # mean_readout = 40, # lab = c("**", "****", "****"), # Exp = as.factor(9) # ) # # ann_text1 <- # data.frame( # Dose = factor (c(0.5, 1, 10), levels = c("0.5", "1", "10")), # mean_readout = 40, # lab1 = c("*", "***", "****"), # Exp = as.factor(10) # ) # # ann_text2 <- # data.frame( # Dose = factor (c(0.25, 0.5, 1, 1.5), levels = c(0.25, 0.5, 1, 1.5)), # mean_readout = 40, # lab2 = c("*", "****", "**", "****"), # Exp = as.factor(14) # ) # # ann_text3 <- # data.frame( # Dose = factor(1), # mean_readout = 40, # lab3 = "*", # Exp = as.factor(15) # ) # # ann_text4 <- # data.frame( # Dose = factor(c(0.5, 1, 2, 3.75), levels = c(0.5, 1, 2, 3.75)), # mean_readout = 40, # lab4 = c("*", "*", "****", "****"), # Exp = as.factor(16) # ) # # bb_dat_plot_nic <- on_plate_stats %>% # filter (Exp == "9" | Exp == "10") %>% # ggplot(aes(x = Dose, y= mean_readout, fill= Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsBB)) + # scale_fill_manual(values=c('#000000','#333333', '#666666','#999999', '#CCCCCC')) + # theme (strip.text.x = element_text(size=12)) + # geom_text(data = ann_text, aes(label = lab)) + # geom_text(data = ann_text1, aes(label = lab1)) + # scale_y_continuous(breaks=seq(0,45,10))+ # ylab("Body bends/min") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # strip.text.x = element_text(size=12), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # # bb_dat_plot_nit <- on_plate_stats %>% # filter (Exp == "11" | Exp == "12") %>% # ggplot(aes(x = Dose, y= mean_readout, fill= Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsBB)) + # scale_fill_manual(values=c('#000000','#339900')) + # theme (strip.text.x = element_text(size=12)) + # scale_y_continuous(breaks=seq(0,45,10))+ # ylab("Body bends/min") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # bb_dat_plot_thia <- on_plate_stats %>% # filter (Exp == "13" | Exp == "14") %>% # ggplot(aes(x = Dose, y= mean_readout, fill= Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsBB)) + # scale_fill_manual(values=c('#000000','#000066', '#0000CC','#0000FF', '#0033FF', '#3366CC')) + # theme (strip.text.x = element_text(size=12)) + # geom_text(data = ann_text2, aes(label = lab2)) + # scale_y_continuous(breaks=seq(0,45,10))+ # ylab("Body bends/min") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # strip.text.x = element_text(size=12), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # bb_dat_plot_clo <- on_plate_stats %>% # filter (Exp == "15" | Exp == "16") %>% # ggplot(aes(x = Dose, y= mean_readout, fill= Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsBB)) + # scale_fill_manual(values=c('#000000','#993300', '#996633','#CC9933', '#FFCC33')) + # theme (strip.text.x = element_text(size=12)) + # geom_text(data = ann_text3, aes(label = lab3)) + # geom_text(data = ann_text4, aes(label = lab4)) + # scale_y_continuous(breaks=seq(0,45,10))+ # ylab("Body bends/min") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # strip.text.x = element_text(size=12), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # bb_grid <- plot_grid(bb_dat_plot_nic, bb_dat_plot_nit, bb_dat_plot_thia, bb_dat_plot_clo, nrow=4) # bb_grid = bb_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) + # ggsave("fig/results2/final/pngpdf/bodybendsplot.pdf")  mk11g11 committed Sep 16, 2019 442 443 444 # clo_grid <- plot_grid(pump_on_food_plot_nic, pump_on_food_plot_nit, pump_on_food_plot_thia, pump_on_food_plot_clo, nrow=4) # clo_grid = clo_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) # clo_grid  mk11g11 committed Feb 19, 2020 445 446 knitr::include_graphics("fig/results2/final/pngpdf/bodybendsplot.png")  mk11g11 committed Sep 16, 2019 447 448   mk11g11 committed Apr 13, 2020 449 (ref:DR-body-bends) **Concentration dependence curves for the effects of nicotine and neonicotinoids on *C. elegans* body bends.** Concentration-response curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on body-bend rates of wild-type N2 and *bus-17* mutant *C. elegans*. Body bend rates are expressed as a % of control activity. Data are mean $\pm$ SEM. The EC~50~ of thiacloprid on N2 and clothianidin on *bus-17* is an approximation, as at highest concentrations tested (1.5 mM thiacloprid and 3.75 mM clothianidin) the maximum inhibition observed was 21 and 42 %.  mk11g11 committed Sep 16, 2019 450   mk11g11 committed Apr 13, 2020 451 {r DR-body-bends-label, fig.cap="(ref:DR-body-bends)", fig.scap= " The concentration dependence curves for the effects of nicotine and neonicotinoids on \\textit{C. elegans} body bends.", fig.width=10, fig.align='center', fig.asp=1.1, echo=FALSE}  mk11g11 committed Sep 16, 2019 452 453 454 455 456 457 458  knitr::include_graphics("fig/results2/DR-body-bends.png")  \newpage #### Effects on egg laying  mk11g11 committed Apr 13, 2020 459 On-plate experiments allow to assay for other aspects of *C. elegans* biology such as egg-laying (Section \@ref(egglayingbehaviour)). The number of eggs laid per worm in the presence of nicotine and neonicotinoids over a period of 24 hours was counted and compared to the control (Figure \@ref(fig:EL-plot-capt-label) and \@ref(fig:egg-laying-lbl)). Both N2 wild-type and *bus-17* mutants lay ~ 95 eggs/day/worm. No effects on wild-type worm of either compound was observed. However, egg-laying rate of *bus-17* mutant was reduced by low mM concentrations of thiacloprid and clothianidin.  mk11g11 committed Sep 16, 2019 460   mk11g11 committed Apr 13, 2020 461 (ref:EL-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoids on egg-laying of *C. elegans*.** Wild type (left panel) and *bus-17* (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0), incorporated into solid medium. Number of eggs laid in 24 hours were counted and expressed as eggs laid per worm. Data are mean $\pm$ SEM collected from $\ge$ 8 individual on $\ge$ 2 days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01, $***$ P $\le$ 0.001.  mk11g11 committed Sep 16, 2019 462   mk11g11 committed Apr 13, 2020 463 {r EL-plot-capt-label, fig.cap= "(ref:EL-plot-capt)", fig.scap = "The concentration dependence for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', fig.asp=1.2, include=TRUE, echo=FALSE}  mk11g11 committed Feb 19, 2020 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 # labelsEL <- c("17" = "Nicotine N2", "18" = "Nicotine bus17", "19"= "Nitenpyram N2", "20" = "Nitenpyram bus17", "21" = "Thiacloprid N2", "22" = "Thiacloprid bus17", "23" = "Clothianidin N2", "24" = "Clothianidin bus17") # ann_textEL <- data.frame(Dose = factor (c(0.25,0.5,1.5), levels = c("0.25", "0.5", "1.5")), mean_readout = 105,labEL = c("*","**","**"), Exp = 22) # # ann_textEL1 <- data.frame(Dose = factor (3.75), mean_readout = 105,labEL1 = "**", Exp = 24) # # el_dat_plot_nic <- on_plate_stats %>% # filter (Exp == "17" | Exp == "18") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEL))+ # theme (strip.text.x = element_text(size=12)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # scale_y_continuous(breaks=seq(0,125,25))+ # scale_fill_manual(values = c('#000000','#333333')) + # ylab("Eggs laid/24hrs/worm") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # strip.text.x = element_text(size=12), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # # el_dat_plot_nit <- on_plate_stats %>% # filter (Exp == "19" | Exp == "20") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEL))+ # theme (strip.text.x = element_text(size=12)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # scale_y_continuous(breaks=seq(0,125,25))+ # ylab("Eggs laid/24hrs/worm") + # scale_fill_manual(values=c('#000000','#339900')) + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # # el_dat_plot_thia <- on_plate_stats %>% # filter (Exp == "21" | Exp == "22") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEL))+ # theme (strip.text.x = element_text(size=12)) + # geom_text(data = ann_textEL, aes(label = labEL)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # scale_y_continuous(breaks=seq(0,125,25))+ # scale_fill_manual(values=c('#000000','#000066', '#0000CC','#0000FF', '#0033FF', '#3366CC', '#66CCFF')) + # ylab("Eggs laid/24hrs/worm") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # # el_dat_plot_clo <- on_plate_stats %>% # filter (Exp == "23" | Exp == "24") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_bar(stat = "identity") + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEL))+ # theme (strip.text.x = element_text(size=12)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_text(data = ann_textEL1, aes(label = labEL1)) + # scale_y_continuous(breaks=seq(0,125,25)) + # scale_fill_manual(values=c('#000000','#993300', '#996633','#CC9933', '#FFCC33')) + # ylab("Eggs laid/24hrs/worm") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # # el_grid <- plot_grid(el_dat_plot_nic, el_dat_plot_nit, el_dat_plot_thia, el_dat_plot_clo, nrow=4) # el_grid = el_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) + # ggsave("fig/results2/final/pngpdf/egglayingplot.pdf") knitr::include_graphics("fig/results2/final/pngpdf/egglayingplot.png")  mk11g11 committed Sep 16, 2019 561 562   mk11g11 committed Feb 19, 2020 563 (ref:DR-egg-laying) **Dose-response curves for the effects of nicotine and neonicotinoids on egg-laying of *C. elegans*.** Concentration-response curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on egg-laying of N2 wild-type and *bus-17* mutant *C. elegans*. Egg laying is expressed as a % control activity. The EC~50~ for clothianidin is an approximation, as at the highest concentration tested (3.75 mM), the maximum response observed was 44 %. Data are mean $\pm$ SEM.  mk11g11 committed Sep 16, 2019 564   mk11g11 committed Apr 13, 2020 565 {r, egg-laying-lbl, fig.cap="(ref:DR-egg-laying)", fig.scap = " Dose-response curves for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 16, 2019 566 567 568 569 570 571 knitr::include_graphics("fig/results2/DR-egg-laying.png")  \newpage #### Effects on egg hatching  mk11g11 committed Feb 19, 2020 572 573 Eggs laid on the plate hatch after 9 hours of exo-utero development (Figure \@ref(fig:life-cycle-label)). To investigate the effects of nicotine and neonicotinoids on egg-hatching L4 + 1 worms were incubated with nicotine and neonicotinoids. After 24 hours of incubation, they were removed from the experimental plate, leaving the progeny and eggs behind. After a further 24 hours, the number of unchanged eggs and larvae present were counted to derive the % hatching rate. Almost 100 % of eggs laid by N2 and *bus-17* hatched (Figure \@ref(fig:EH-plot-label)). Neither compound had an effect on hatching of N2 worms (Figure \@ref(fig:EH-plot-label), left panel). However, thiacloprid at 1.5 mM and clothianidin at 2 mM reduced the proportion of hatched eggs of *bus-17* worms by 19 and 13 %, respectively (Figure \@ref(fig:EH-plot-label), right panel).  mk11g11 committed Apr 13, 2020 574 (ref:EH-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoids on *C. elegans* egg-hatching.** N2 wild-type (a) and *bus-17* mutant (b) worms laid eggs in the presence of varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0). After 24 hours adult worms were removed and the eggs left behind. Number of unhatched eggs and larvae were counted 24 hours later. Data are mean $\pm$ SEM, of $\ge$ 2 paired experiments performed on $\ge$ days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01.  mk11g11 committed Feb 19, 2020 575   mk11g11 committed Apr 13, 2020 576 {r EH-plot-label, fig.cap = "(ref:EH-plot-capt)", fig.align='center', fig.cap= "(ref:EH-plot-capt)", fig.scap = " The concentration dependence for the effects of nicotine and neonicotinoids on \\textit{C. elegans} egg-hatching.", echo=FALSE}  mk11g11 committed Feb 19, 2020 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 # labelsEH <- c("25" = "Nicotine N2", "26" = "Nicotine bus17", "27"= "Nitenpyram N2", "28" = "Nitenpyram bus17", "29" = "Thiacloprid N2", "30" = "Thiacloprid bus17", "31" = "Clothianidin N2", "32" = "Clothianidin bus17") # ann_textEH <- data.frame(Dose = factor (c(1, 1.5), levels=c("1", "1.5")), mean_readout = 105,labelEH = c("*","**"), Exp = 30) # # ann_textEH1 <- data.frame(Dose= factor(2), mean_readout = 105, labelEH1 = "*", Exp=32) # # eh_dat_plot_nic <- on_plate_stats %>% # filter (Exp == "25" | Exp== "26") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_bar(stat = "identity") + # scale_fill_manual(values = c('#000000','#333333')) + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEH))+ # theme (strip.text.x = element_text(size=12)) + # ylim(0, 120) + # ylab("% eggs hatched") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # panel.grid.minor = element_blank(), # panel.background = element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # eh_dat_plot_nit <- on_plate_stats %>% # filter (Exp == "27" | Exp== "28") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_bar(stat = "identity") + # scale_fill_manual(values = c('#000000','#339900')) + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEH))+ # theme (strip.text.x = element_text(size=12)) + # ylim(0, 120) + # ylab("% eggs hatched") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # eh_dat_plot_thia <- on_plate_stats %>% # filter (Exp == "29" | Exp== "30") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_bar(stat = "identity") + # geom_text (data = ann_textEH, aes(label = labelEH)) + # scale_fill_manual(values=c('#000000','#000066', '#0000CC','#0000FF', '#0033FF', '#3366CC')) + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEH))+ # theme (strip.text.x = element_text(size=12)) + # ylim(0, 120) + # ylab("% eggs hatched") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # eh_dat_plot_clo <- on_plate_stats %>% # filter (Exp == "31" | Exp== "32") %>% # ggplot(aes(x = Dose, y= mean_readout, fill=Dose)) + # geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) + # geom_bar(stat = "identity") + # geom_text (data = ann_textEH1, aes(label=labelEH1)) + # scale_fill_manual(values=c('#000000','#993300', '#996633','#CC9933', '#FFCC33','#FFCC66')) + # theme(legend.position="none") + # facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labelsEH))+ # theme (strip.text.x = element_text(size=12)) + # ylim(0, 120) + # ylab("% eggs hatched") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) # # eh_grid <- plot_grid(eh_dat_plot_nic, eh_dat_plot_nit, eh_dat_plot_thia, eh_dat_plot_clo, nrow = 4) # eh_grid = eh_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) + # ggsave("fig/results2/final/pngpdf/egghatchingplot.pdf") knitr::include_graphics("fig/results2/final/pngpdf/egghatchingplot.png")  mk11g11 committed Sep 16, 2019 670 671 672 673 674 675  \newpage To investigate whether compounds hinder the hatching of larvae, images of unhatched eggs were taken. As seen in (Figure \@ref(fig:unhatched-eggs-labels)), the eggs laid in the presence of thiacloprid and clothianidin are granular in appearance with no worm inside. This suggests thiacloprid and clothianidin interfere with the process of fertilisation or early developmental processes.  mk11g11 committed Feb 19, 2020 676 (ref:unhatched-eggs) **Effects of thiacloprid and clothianidin on *C. elegans* egg-hatching.** The appearance of unhatched eggs laid by *bus-17 C. elegans* mutant in the presence of 1.5 mM thiacloprid.  mk11g11 committed Sep 16, 2019 677   mk11g11 committed Apr 13, 2020 678 {r unhatched-eggs-labels, fig.cap="(ref:unhatched-eggs)", fig.scap= " Effects of thiacloprid and clothianidin on \\textit{C. elegans} egg-hatching.", fig.align='center', echo=FALSE}  mk11g11 committed Feb 19, 2020 679 knitr::include_graphics("fig/results2/unhatched-egg-2.png")  mk11g11 committed Sep 16, 2019 680 681 682 683 684  \newpage #### Effects on development  mk11g11 committed Feb 19, 2020 685 Eggs that are laid on plate with a food source, hatch and develop into adults in three days. During the on-plate assay an observation was made that there were smaller worms present on the plate containing nicotine and thiacloprid (Figure \@ref(fig:dev-image)). To investigate whether this was an effect of the drugs on the timing of development, larval development of age-synchronized progeny was made.  mk11g11 committed Sep 16, 2019 686 687 688  (ref:development-images-capt) **Effects of nicotine and thiacloprid on larval development of *C. elegans*.** Eggs were laid by N2 wild-type worms on medium containing 1 mM nicotine or 1 mM thiacloprid. 72-hours later, the images of the progeny were taken. Worms developing in the presence of treatment are visibly smaller in comparison to the control.  mk11g11 committed Apr 13, 2020 689 {r dev-image, fig.cap= "(ref:development-images-capt)", fig.scap = " Effects of nicotine and thiacloprid on larval development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 16, 2019 690 691 692 693 694 knitr::include_graphics("fig/results2/Development_images.png")  \newpage  mk11g11 committed Feb 19, 2020 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 A synchronous population of L4 + 1 worms laid eggs on drug-treated plate. 2 hours later, the adults were removed from the plate. The development of the progeny was observed. The number of worms in each developmental stage, namely L1, L2, L3 and L4 was made at days 1, 2, 3 and 6 days post egg laying. Clothianidin or nitenpyram at 1mM showed no effect as the proportion of each developmental stage shifted in parallel with N2 (data not shown). In contrast thiacloprid and nicotine slowed the larval development of worms (Figure \@ref(fig:development-selected-plot)). This difference was most clearly observed at day two. Almost all control worms reached L3 stage in control plate. 50 % of thiacloprid exposed worms were L3 and the rest were still at the L2 stage. Nicotine had a greater effect with almost all the worms being L2. This suggests L2/L3 transition was disturbed. All worms reached adulthood by day 6 of their life.  mk11g11 committed Sep 16, 2019 730 731 732  (ref:development-plot-capt) **Effects of nicotine and thiacloprid on the development of *C. elegans*.** N2 wild-type worms laid eggs on plates dosed with 1mM thiacloprid, 1mM nicotine or drug vehicle (Ctr). Larval development in the presence of drugs was monitored over time. Worms were assigned to each one of 5 life-stages, namely L1, L2, L3, L4 and gravid adults. The fraction of worms in each stage as a % of total population at time point: 30, (day 1), 48 hours (day 2), 72 hours (day 3), 144 hours (day 6) was measured. Data are shown as the mean of N $\ge$ 3.  mk11g11 committed Apr 13, 2020 733 {r development-selected-plot, fig.cap= "(ref:development-plot-capt)", fig.scap= " Effects of nicotine and thiacloprid on the development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 16, 2019 734 #select values  mk11g11 committed Feb 19, 2020 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 # dev_t3 <- dev_dat_t2 %>% # filter(Comp == "Nic" | Comp == "Thia" | Comp == "Ctr") %>% # filter(Time == 30 | Time == 48 | Time == 72 | Time == 144) # # cbp1 <- c("#999999", "#E69F00", "#56B4E9", "#009E73", # "#F0E442", "#0072B2", "#D55E00", "#CC79A7") # # sel_dev_plot <- dev_t3 %>% # group_by (Time, Comp, Stage) %>% # ggplot(aes(x= Comp, y= Worms, fill= Stage)) + # facet_wrap(~Time) + # geom_bar(stat = "identity") + # scale_fill_manual(values = cbp1) + # ylab ("% worms") + # xlab("Treatment") + # labs(fill = "Developmental stage") + # theme(axis.text = element_text(size=12), # axis.title.x=element_blank(), # axis.title = element_text(size=12), # strip.text.x = element_text(size=12), # panel.background = element_blank(), # axis.line = element_line(colour = "black"), # plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), # text = element_text(size=12, family="sans")) + # ggsave("fig/results2/final/pngpdf/developmentplot.pdf") knitr::include_graphics("fig/results2/final/pngpdf/developmentplot.png")  mk11g11 committed Sep 16, 2019 762 763 764 765 766  \newpage ## Discussion  mk11g11 committed Feb 19, 2020 767 768 769 Investigation of the environmental safety profile of pest controlling compounds is essential to ensure safe use of these substances [@iyaniwura1991]. Neonicotinoids are the most commonly used insecticides worldwide, but their impact on many non-target invertebrates is poorly understood. To determine their potential environmental impact, the effects of neonicotinoids on the Nematoda representative *C. elegans* has been investigated. Limited number of studies have investigated the effects of neonicotinoids on *C. elegans*. These investigations typically report disruption of behaviors governed by cholinergic neurotransmission [@gomez-eyles2009; @ruan2009; @mugova2018; @hopewell2017]. In this chapter detailed description of the effects of neonicotinoids on various aspects of worm behavior are described and compared to the effects exerted by a classical nicotinic acetylcholine receptor agonist, nicotine.  mk11g11 committed Sep 16, 2019 770 771  ### Nicotine and neonicotinoids affect locomotion of worms by differential mechanisms  mk11g11 committed Feb 19, 2020 772 Wild-type animals were exposed acutely to nicotine and neonicotinoids and their effects on thrashing a measure of motility was scored. Neither thiacloprid nor clothianidin impaired motility. In contrast, nitenpyram and nicotine paralysed worms when present at mM concentrations. Increase in exposure time from 2 to 24 hours resulted in increased efficacy of nicotine on wild-type worms in motility assay which now utilized body bends. This was reflected in the shift of EC~50~ from 31 to 3.6 mM. Thiacloprid had no effect on thrashing, but it inhibited body bends by 20 % at 1.5 mM. Clothianidin had no effect on thrashing or body bends.  mk11g11 committed Sep 16, 2019 773   mk11g11 committed Feb 19, 2020 774 Despite common effects of nicotine and neonicotinoids on locomotion, the mode of action is likely to be different. Exposure of worms to nicotine for 4 hours led to significant reduction in the size of the worm. This is due to the proposed mode of action of nicotine. Nicotine activates ACR-16 nicotinic acetylcholine receptors present at the body wall muscle [@touroutine2005; @richmond1999], which leads to BWM hypercontraction [@sobkowiak2011]. The BWM is physically attached to the cuticle [@altun2009], therefore prolonged muscle stiffness and hypercontraction can lead to shrinkage of the cuticle [@petzold2011] and the reduction in the body size was observed in this study. In contrast, neonicotinoids did not have the same effect. Thiacloprid and clothianidin treated worms showed uncoordinated, twitchy phenotype and no reduction in the body length. Taken together, this data suggest that nicotine and neonicotinoids can act to inhibit locomotion differently. It is possible that they all act on nAChRs in the worm, as these molecules are the primary targets for nicotine and neonicotinoids in other species, but they are likely to act on different types of *C. elegans* nAChRs. To provide an insight into the mode of action of these compounds, studies on wild-type and nAChR mutant worms should be performed. Behavioral analysis of wild-type and mutant strains exposed to nicotine and neonicotinoids might allow for identification of strains resistant to these compounds and hence discover potential molecular targets.  mk11g11 committed Sep 16, 2019 775 776  ### Nicotine inhibition of thrashing  mk11g11 committed Oct 06, 2019 777   mk11g11 committed Feb 19, 2020 778 The time course for the effects of 10 and 25 mM nicotine on thrashing of wild-type worm revealed two “phases” of inhibition. First, seen after 10 minutes, followed by partial recovery and a second phase seen after 40 minutes. This could suggest nicotine targets multiple sites that alter thrashing. Previous research has shown that nicotine acts at a body wall muscle but also at sensory neurons. Nicotine is an agonist of TRP $\beta$ receptors [@feng2006] which are expressed in nociceptive ASH and ADL neurons [@colbert1997]. These neurons send out processes to the nerve ring where they make connections with a diversity of circuits, including those regulating movement [@rogers2006]. A nicotine-related compound quinine has an effect on locomotion via nociceptive circuit [@hilliard2004]. It is therefore possible that the first and rapid effect of nicotine on thrashing could be due to the action on sensory neurons. Sensory neurons have processes exposed to the external environment [@hall2008], which are easily accessible to nicotine. Indeed, nicotine at high doses inhibits locomotion within 1 minute of incubation (Figure \@ref(fig:onset-plot-label)a). In addition, this effect is equally rapid on wild-type and *bus-17* mutant worms. This suports the notion that nicotine affects locomotion by targeting structures exposed to the surface and not buried within the worm.  mk11g11 committed Sep 16, 2019 779   mk11g11 committed Feb 19, 2020 780 The second phase of inhibition could be due to the effects of nicotine on the body wall muscle. It may take longer to reach muscular targets because worms ingest only a little material whilst in liquid [@gomez-amaro2015] and so nicotine must cross the cuticle to reach the BWM. The complex structure and the thickness of the cuticle may slow the kinetics of absorption. This is supported by the lack of two phases of nicotine-induced paralysis in the leaky cuticle *bus-17* mutant. The effects of nicotine on thrashing of a mutant strain equilibrate after 10 minutes of incubation which may suggest improved permeability and hence reflect synergistic sensory and muscular effects of nicotine on locomotion.  mk11g11 committed Sep 16, 2019 781   mk11g11 committed Feb 19, 2020 782 783 ### The cuticle limits bioavailability of nicotine and neonicotinoids This study reports low efficacy of neonicotinoids on the wild-type worm *C. elegans* (Table \@ref(tab:discuss1-summary-table)). This could be due to a low potency of compounds on target receptors, or due to the limited permeability. The increased efficacy of nicotine on wild-type worm motility in terms of body bend vs thrashing assay suggest that nicotine does not equilibrate across the cuticle readily. This suggest that low *C. elegans* sensitivity to nicotine (and potentially neonicotinoids) might be a result of hindered permeability of drugs.  mk11g11 committed Sep 16, 2019 784   mk11g11 committed Feb 19, 2020 785 Permeability of drugs across lipidic structures is dependent on the physicochemical properties of compounds [@avdeef2004]. The effects of the pH of the external buffer on the efficacy of nicotine on thrashing were investigated. The changes in pH shifts the ionization state of nicotine, which could affect absorption. A shift of pH from 7 to 6 and 9 moderately decrease EC~50~ from 26.2 mM to 16.7 mM and 15.2 mM, respectively. Therefore the pH of external solution does not markedly alter efficacy of nicotine. This may be due to worms’ capacity to regulate their cuticular surface pH in tunnel-like and water-filled pores. These structure are on the surface of the cuticle and keep constant pH microenvironment of ~5 [@sims1992; @sims1994]. There are other physicochemical factors that could play a role in permeability of nicotine and neonicotinoids, such as lipophilicity. Nicotine and neonicotinoids have moderate lipophilicity [@blaxten1993] which could limit the ability of drugs to enter and diffuse across lipidic structures [@liu2011], limiting bioavailibity and efficacy of compounds. If the primary molecular targets are within and not on the surface of the worm, the low efficacy could be due to the limited diffusion through the cuticular structures.  mk11g11 committed Sep 16, 2019 786 787 788 789  To investigate the role of surface coat and cuticle in drug permeability *bus-17* mutant with fragile and more permeable cuticle was employed in acute and moderate exposure assays. Exposure of *bus-17* worms increased potency of all compounds. For example, in acute-exposure, thrashing experiments, the EC~50~ for the effects of nicotine and nitenpyram increased by 8- and 5-fold, respectively. Moreover, thiacloprid and clothianidin had no effect on motility of the wild-type worm, but on *bus-17* they induced paralysis with the EC~50~ of 480 $\mu$M and 2.2 mM, respectively. Increased drug sensitivity was also observed in other cuticle-mutant strains for example *bus-8* [@partridge2008], *bus-5* and *bus-16* [@xiong2017]. This increased sensitivity is thought to be due to an increased cuticular permeability in mutant strains [@partridge2008; @xiong2017]. These data highlights the importance of the cuticle in protecting *C. elegans* against the outer environment. The cuticle is a common structural feature of all soil nematodes [@decraemer2003]. It is likely that it limits bioavailability of residual insecticides in all soil nematodes, hindering their potential toxic effects. #### Nicotine and thiacloprid have a neurodevelopmental effect on *C. elegans*.  mk11g11 committed Oct 06, 2019 790   mk11g11 committed Sep 16, 2019 791 792 Worms growing in the presence of 1 mM nicotine and thiacloprid developed into adults slower than the control worms. Neonicotinoids also disrupt larval development in bees [@souzarosa2016]. In would be interesting to investigate whether there is a common mechanism of neonicotinoid-induced neurodevelopmental defect in worms and bees. In worms, thiacloprid acts by disrupting L2/L3 transition. L2 stage is a stage at which multiple cell divisions and differentiation occurs [@hall2008]. Nicotinic acetylcholine receptors containing UNC-63 subunits seem to be involved in this process [@ruaud2006]. Developmental assays on *unc-63* and other nAChR mutants should be carried out to determine if the action of thiacloprid depends on this protein.  mk11g11 committed Oct 06, 2019 793 794 \newpage  mk11g11 committed Sep 16, 2019 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 ------------------------------------------------------ Behavioral Compound Strain EC~50~ assay -------------- ---------------- --------- ------------ Thrashing Nicotine N2 26.2mM *bus-17* 3.3mM Nitenpyram N2 195.8mM *bus-17* 16.6mM Thiacloprid N2 > 1.5mM *bus-17* 377.6$\mu$M Clothianidin N2 > 2.5mM *bus-17* 3.3mM Body bends Nicotine N2 3.6mM *bus-17* 1.6mM Nitenpyram N2 > 1mM *bus-17* > 1 mM Thiacloprid N2 3.7mM *bus-17* 721.2$\mu$M Clothianidin N2 > 3.75mM *bus-17* 3.3mM Egg-laying Nicotine N2 > 1mM *bus-17* > 1mM Nitenpyram N2 > 1mM *bus-17* > 1mM Thiacloprid N2 > 1mM *bus-17* 1.4mM Clothianidin N2 > 3.75mM *bus-17* 6.2mM Egg-hatching Nicotine N2 > 1mM *bus-17* > 1mM Nitenpyram N2 > 1mM *bus-17* > 1mM Thiacloprid N2 > 1mM *bus-17* 1.5mM Clothianidin N2 > 3.75mM *bus-17* > 3.75mM ------------------------------------------------------ Table: (\#tab:discuss1-summary-table) Summary table of the effects of nicotine and neonicotinoids on *C. elegans*.