03-results-01.Rmd 78.1 KB
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# Effects of neonicotinoids on the behaviour and development of *C. elegans* {#results-1}
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```{r echo=FALSE, include=FALSE}
library(cowplot)
library(tidyverse)
library(ggpubr)
library(readr)
library(ggplot2)
library(scales)
library(curl)
library(devtools)
library(knitr)
```

## Introduction
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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. 
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## Ecological role of non-parasitic worms 

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 assessment of contaminated soil [@lecomte-pradines2014]. 
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### Residues of neonicotinoids in soil 
Neonicotinoids are commonly applied as a seed dressing [@jeschke2011; @alford2017], due to a benefit of extended crops protection resulting in a reduction of 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. 

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 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 sorption capacity means that neonicotinoids may come in contact with soil- residing worms for prolonged time periods. Neonicotinoids can also enter 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.

<!-- ### **C. elegans** in toxicity testing -->
<!-- *C. elegans* is a simple organism, easy to maintain, with well defined anatomy (Sections @\ref(anatomy)) and behaviour (Section @\ref(analytical_behaviour)). One of its major advantages is that it amenable to genetic manipulations (Section @\ref(genmanip)). Since its isolation in 1950, it has grown into an alternative model for the mode of action and toxicity studies of many compounds, most notably antiparasites [@Holden-Dye2014] such as levamisole, as well as an insect repelent N,N-diethyl-meta-toluamide (DEET). *C. elegans* is an attractive platform for the toxicity and the mode of action studies but it may be also useful in drug development screens. Several studies showed that the lethality rank order of hundreds of comounds tested on *C. elegans* correlated well with the lethality rank order in other species [reviewe in @hunt2017].  -->

<!-- One of the limitations of *C. elegans* is their cuticle (Section @\ref(anatomy)). The cuticle is a robost structure, composed primarely from cross-links of collagen. It can limit entry of drugs present in the extenral environment, hindering their bioavailability. To this end, several strains have been identified as "leaky". These strains have mutated bus (Bacterially UnSwollen) genes. To date six such genes have been cloned: bus-2, bus-4, bus-8, bus-12, bus-17 and srf-3. Bus-2, 4 and 17 encode for glycosyltranferases [@gravato-nobre2011]. Bus-8 mannosyltransferase [@partridge2008], whereas bus-8 [@gravato-nobre2011] and srf-3 [@hoflich2004] nucleotide sugar transporters. Bus mutants are characterised by altered surface protein coat, resulting in reduced immune response, cuticle integrity and strength. This in turn leads to blunted pathogen entry and evasion [@gravato-nobre2005; @cipollo2004; @hoflich2004], compromised mechanical defence [@darby2007], as well as markedly increased drug-sensitivity [@partridge2008], abnormal locomotion (skiddy phenotype with low traction [@darby2007] and delayed development [@gravato-nobre2005]. Due to the increased cuticular permeability, **bus** mutants have been suggested an alternative and approprite platform for toxicity studies of pharmaceuticals [@xiong2017]. -->

### Cholinergic regulation of worm behaviour ###{#cholinericwormbeh}
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 soil nematode and model organism *C.elegans*.

#### Locomotion ####{#locomotion}
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*C. elegans* exhibits distinct locomotory behaviours in liquid and on the 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)). 
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There are 4 motor neuron classes synapsing onto dorsal muscle (AS, DA, DB, and DD) and 4 innervating 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]. Bend of 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 worm’s locomotory activities. Whereas the propagation of the electrical signal down the axis of the muscle whilst on solid medium, results in the forward movement. 

##### Regulation of locomotory behaviour
The rhythmical pattern of muscular contraction and relaxation of the BWM is not regulated by the nervous system. Worms lacking functional D neurons are still capable of classical movement behavior [@riddle1997b]. Instead, body stretch associated with movement is detected. This results in electrical signal propagation to further segments of the BWM and coordinated movement [@tavernarakis1997]. According to this model, the bend of one segment of the musculature is detected by the proprioceptive ion channels on the adjecent motor neuron. The activation of this leads to a strong contraction of the next muscle in the series reflected in a body bend. This pattern is repeated which leads to a propagation of the signal down the body length. 

##### Regulation of the direction of movement
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]. 

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#### Sensory regulation of the locomotion ####{#sensoryregulation}
Locomotion can be regulated by the environmental cues detected by the sensory neurons which relay information into 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 enahnced 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 of food evident by enahnced 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). 
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(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")
```

#### Egg laying
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 serotonin [@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]. 

(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',}
knitr::include_graphics("fig/intro_2/vulva.jpg")
```

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#### *C. elegans* nAChRs ####{#muscletypenachr} 
The identity of two muscle-type nAChRs was depicted in a combination of behavioural, pharmacological and electrophysiological approaches. 

##### L-type receptors
Behavioural analysis of *C. elegans* mutants identified several strains in which locomotion was disrupted, including unc-29, unc-38 and unc-63 [@lewis1980b]. unc-29, unc-38 and unc-63, as well as lev-1 and lev-8 were also resistant to levamisole [@lewis1980b]. Expression of lev-1, unc-29 and unc-38 in Xenopus oocytes generated a protein with nAChR-like properties: in response to acetylcholine and levamisole, depolarising current was elicited [@fleming1997]. @richmond1999 provided evidence that these receptors are expressed at the NMJ of the body wall muscle. Intracellular recordings from the post-synaptic membrane at the NMJ of the body wall muscle showed that in response to acetylcholine and levamisole inward current is elicited. That current was abolished in unc-29 and unc-38 mutants [@richmond1999]. The identity of the levamisole sensitive nAChRs was revealed by @boulin2008, who showed that eight genes are required for the generation of fully functional receptor in Xenopus oocytes. Five genes encode for nAChR subunits UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8, two of which, viz. UNC-29 and LEV-1 are non-$\alpha$. In the absence of any one of the 5 subunits, agonist-evoked currents were abolished, suggesting all subunits are essential for the receptor function. The remaining 3 genes encode for the auxiliary subunits RIC-3, UNC-50, AND UNC-74. Their role is described in Section \@ref(cematnachr). 

##### N-type receptors 
Work of @richmond1999 identified second type of nAChR at the muscular junction of the body wall muscle. This receptor showed high sensitivity to nicotine, thus was named N-type nAChR. N-type receptor is composed of ACR-16 subunits, which form homomeric receptors in Xenopus oocytes [@ballivet1996].

### Effects of nAChR agonists on *C. elegans* behaviour ###{#pharmacelegans}
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Pharmacological experiments in which nAChR agonists were tested against *C. elegans* behaviours provide evidence for the important role of these receptors in the regulation of locomotion and egg laying. 

#### Levamisole
Levamisole is a synthetic compound used in treatment of parasitic worm infestation in both humans and animals [@miller1980]. It is an agonist of a subset of receptors present at a body wall muscle [@richmond1999]. Levamisole causes spastic paralysis of worms [@lewis1980b] and stimulates egg-laying [@trent1983].

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#### Nicotine 
Nicotine is an alkaloid naturally occuring in the Tobacco plant [@steppuhn2004]. It is an agonist of the second type receptor at a body wall muscle, namely the N-type [@], but based on the nicotine-intoxication worm phenotype, it is likely to target receptors regulating pharyngeal pumping and vulva muscle. Nicotine inhibits locomotion [@kudelska2017] pharyngeal pumping [@kudelska2018], and egg-laying.
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<!-- ### Effects of neonicotinoids on earthworms -->

<!-- Most ecotoxicological studies focused on the effects of neonicotinoids on behaviours governed by the cholinergic neurotransmission and worms mortility. These were conducted on earthworms, Lumbricus terrestris and Eisenia fetida, which reflects the pivotoal ecological role of these “undrgrdound dwellers”. @basley2017 showed that field realistic concentrations of clothianidin have no effects on Lumbricus terrestris mortality, feeding and worm population [@basley2017], but it is toxic to Eisenia fetida with LC50 value of ~1 mM [@wang2012]. -->

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#### Neonicotinoids ####{#chapter3effectsofneonics}
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<!-- There is a limited literature regarding the effects of neonicotinoids on nematodes. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of neonicotinoids. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014]  -->

<!-- Thiaclopand inhibits its egg hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. -->
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Neonicotinoids 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]. 
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<!-- DEET was discovered in 1944 and was originally intendeed for use in agriculture. It is currently used as an insect repellent effective agains flies, mosquitos, ticks and fleas.  -->

<!-- Levamisole, discovered in 1960s, has a potent antihelmintic action [@pinnock1988]. It causes contraction of the body wall muscle of the parasitic worm **A.suum** [@martin1991] and nematode **C. elegans** [@lewis1980b], which leads to their paralysis. Body wall muscle of both **A.suum** and **C. elegans** express N- and L-type nAChR types [@qian2006; @richmond1999]. Genetic studies shed light on the receptor type targeted by levamisole. *C. elegans* mutants deficient in several constituents of L-type, and not N-type nAChR, exihibited markedly reduced levamisole-sensitivity [@lewis1987; @lewis1980], suggesting L-type receptor is the principal site of action of this compound.  -->

<!-- Several reports of of insect strains resistant to DEET have been identified [@reeder2001; @klun2004]. Understanding the mode of action and identification of the molecular target would open the door of opportunities for the development of novel repellents. DEET has a common mode of action, whereby it impairs the olfactory responses to common olfactory attractants and/or repellents in both *C. elegans* [@dennis2018] and Drosophila [@pellegrino2011], albeit by targeting different proteins. In *C. elegans*, sensitivity to DEET is driven by the **str-217** gene encoding for a predicted G protein-coupled receptor, which is not present in insects [@dennis2018].  -->

<!-- Strains useful - When challenged with M. nematophilum, wild-type worm develops characteristic swelling in the tail region. This phenotype is absent in worms with mutated bus (Bacterially UnSwollen) genes.  -->

### 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

### Effects on thrashing
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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. 
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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}
knitr::include_graphics("fig/results2/final/pngpdf/Nic-DR-thrashing-n2_pH.png")
```

\newpage

### Effects of the cuticle on nicotine induced inhibition of thrashing
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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/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.
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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.

```{r thrashing-cuticle-label, fig.cap= "(ref:thrashing-cuticle)", fig.scap = "The effects of the cucticle on the concentration and time dependence of nicotine inhibition of \\textit{C. elegans} thrashing.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/final/pngpdf/fig2.png")
```

\newpage

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### Effects of neonicotinoids on thrashing ###{#effectsofneonicsonthrashing}
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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.
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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].
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(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
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 t~1/2~ (time taken to paralyse 50 % of worms) 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. 

(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

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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 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 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 1/2 of 1.5 hours and 50 minutes respectively. 
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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. 

(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, 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.

```{r merged-recovery-title, fig.cap= "(ref:recovery-thrashing)", fig.scap= "Recovery kinetic of nicotine and nonicotinoid-induced-paralysed \\textit{C. elegans}.", fig.align='center', echo=FALSE}

knitr::include_graphics("fig/results2/final/pngpdf/recovery.png")
```

\newpage

#### Effects on morphology
During acute and chronic exposure experiments, it was noted that 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. 

(ref:shrinking-image) **Effects of nicotine on *C. elegans* morphology.** Images of *bus-17* mutant worms exposed for 4 hours to 25 mM nicotine nicotine, or vehicle.

```{r shrinking-title2, fig.cap="(ref:shrinking-image)", fig.scap = "Effects of nicotine on \\textit{C. elegans} morphology.", fig.align='center',include=TRUE, results="hide", echo=FALSE}
 knitr::include_graphics("fig/results2/Shrinking_nicotine_bus17-2.png")
```

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 body's’ length were made . Exposure to 25 mM nicotine led to reduction in the length of the body size. In contrast, neonicotinoids had no effect (Figure \@ref(fig:shrinking-title1)).

(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.

```{r shrinking-title1, fig.cap="(ref:shrinking)", fig.scap = "Effects of nicotinic compounds on \\textit{C. elegans} body lenght.", include="TRUE", results="hide", fig.align='center', warning=FALSE, echo=FALSE, message=FALSE}

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", "Nicotine_25mM", "Nitenpyram_50mM", "Thiacloprid_1.5mM", "Clothianidin_2.5mM"), 
         labels = c("Ctr", "Nicotine 25mM", "Nitenpyram 50mM", "Thiacloprid 1.5mM", "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(),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          axis.ticks.x = element_blank(),
          axis.title.x = element_blank()) 
shrinking_plot
```

\newpage

### Effects of chronic exposure on behaviour
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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 ranging from 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. 
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#### 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). 

```{r echo=FALSE, include=FALSE, message=FALSE, include=TRUE, results="hide"}
on_plate_dat_1 <-readRDS("Analysis/Data/Transformed/combined.RSD")
on_plate_dat_trans_1 <- on_plate_dat_1 %>% 
  drop_na() %>% 
  mutate(Dose = factor(Conc,
                       levels= c(0, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3.75, 5, 10, 25, 50, 100),
                       labels = c("0", "0.001", "0.01", "0.1", "0.25", "0.5", "0.75", "1", "1.5", "2", "2.5", "3.75", "5", "10", "25", "50", "100")),
                Exp = factor(Experiment,
                           levels= Experiment,
                           labels = Experiment))
```

(ref:avoid) **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.

```{r avoid-label, fig.cap="(ref:avoid)", fig.scap= "Concentration dependence of the effects of nicotine on \\textit{C. elegans} avoidance.", fig.align='center', echo=FALSE}

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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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           axis.title = element_text(size=12),
           text = element_text(size=12, family="sans"))
  avoid
```

\newpage

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#### Effects on body bends ####{#bodybendsneonics}
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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)). 
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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)).

```{r load-stats-on-plate-assay-label, include=TRUE, results="hide", echo=FALSE}
#select bb data 

on_plate_dat <- readRDS("Analysis/Data/Transformed/24_hrs_combined.RDS")

on_plate_dat_trans <- on_plate_dat %>% 
  drop_na() %>% 
  mutate(Dose = factor(Conc,
                       levels= c(0, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3.75, 5, 10, 25, 50, 100),
                       labels = c("0", "0.001", "0.01", "0.1", "0.25", "0.5", "0.75", "1", "1.5", "2", "2.5", "3.75", "5", "10", "25", "50", "100")),
         #Col = factor(Color,
         #levels=Color,
         #labels=Color),
                Exp = factor(Experiment,
                           levels= Experiment,
                           labels = Experiment))

levels(on_plate_dat_trans$Dose)

on_plate_stats <- on_plate_dat_trans %>% 
  #filter(exp_set=="on_plate_assay") %>% 
  group_by(Assay, Exp, Strain, Comp, Dose) %>% 
  summarise(mean_readout=mean(readout),
            n=n(),
            sd=sd(readout),
            se=sd/sqrt(length(readout)))

```

(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.

```{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.asp=1.2, fig.align='center', include=TRUE, results="hide", echo=FALSE}
#Plot body bends graphs 
#make a data frame for facet labels
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")
#labels for sagnificance to be ploted on a graph 
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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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"))
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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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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"))
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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)
bb_grid
# 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
```

(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 %.

```{r DR-body-bends-label, fig.cap="(ref:DR-body-bends)", fig.scap= "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}

knitr::include_graphics("fig/results2/DR-body-bends.png")
```

\newpage

#### Effects on egg laying
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On-plate experiments allow to assay for other aspects of *C. elegans* biology such as egg-laying (Section \@ref(gglayingbehaviour)). 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-label)) and \@ref(fig:egg-laying-lbl)). Both N2 wild-type and *bus-17* mutants lay ~ 95 egg/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.
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(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 Dunnet’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01, $***$ P $\le$ 0.001, $****$ P $\le$ 0.0001.

```{r EL-plot-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, results="hide", echo=FALSE}
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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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)
el_grid
```

(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~ 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.

```{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}
knitr::include_graphics("fig/results2/DR-egg-laying.png")
```

\newpage

#### Effects on egg hatching
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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-hour incubation, they were removed from the experimental plate, leaving the progeny and eggs behind. After 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)). 
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(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 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 Dunn’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01, $***$ P $\le$ 0.001, $****$ P $\le$ 0.0001.

```{r EH-plot-label, fig.cap = "(ref:EH-plot-capt)", fig.align='center', include=TRUE, results="hide", fig.asp=1.2, echo=FALSE, cache = T, fig.cap= "(ref:EH-plot-capt)", fig.scap = "The concentration-dependence for the effects of nicotine and neonicotinoids on \\textit{C. elegans} egg-hatching."}
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(),
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          panel.grid.minor = element_blank(),
          panel.background = element_blank(),
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          axis.title = element_text(size=12),
          strip.text.x = element_text(size=12),
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          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          plot.margin = unit(c(5.5,5.5,12,5.5), "pt"),
          text = element_text(size=12, family="sans"))

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eh_grid <-  plot_grid(eh_dat_plot_nic, eh_dat_plot_nit, eh_dat_plot_thia, eh_dat_plot_clo, nrow = 4)
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eh_grid = eh_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0)
eh_grid
```

\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.

(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.

```{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}
knitr::include_graphics("fig/results2/unhatched-egg.png")
```

\newpage

#### Effects on development 
Eggs laid on plate with food source, hatch and develop into adults in three days. During on-plate assay an observation of smaller worms present on the plate containing nicotine and thiacloprid was made (Figure \@ref(fig:dev-image)). To investigate whether this was due to an effect on development timing, larval development of age-synchronized progeny was monitored. 

(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.

```{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}
knitr::include_graphics("fig/results2/Development_images.png")
```

\newpage

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 was still at the L2 stage. Nicotine had a greater effect with almost all worms being L2. This suggests L2/L3 transition was disturbed. All worms reached adulthood by day 6 of their life.

```{r development-appendix-plot, fig.cap="(ref:development-plot-capt-2)", include="TRUE", results="hide", echo=FALSE}
# read data in, drop na, transform 
dev_dat <- read_csv("Analysis/Data/Exported/Development.csv",
                    col_types = cols())
#drop_na(dev_dat)
#t(dev_dat)

# transform by gatehring, separate columns 
dev_dat_t <- dev_dat %>%
  gather(key = Stage, value = Worms, -Time,-Cond) %>%
  drop_na() %>%
  separate(Cond, into= c("Comp", "Conc"), sep="_")

#muatate charcaters to factors 
dev_dat_t2 <- dev_dat_t %>%
  mutate(Stage = factor(Stage,
                        levels = c("L1", "L2", "L3", "L4", "Adult"),
                        labels = c("L1", "L2", "L3", "L4", "Adult")),
         Comp = factor(Comp,
                        levels = c("Ctr", "Nic","Thia", "Nit", "Clo"),
                        labels = c("Ctr", "Nic", "Thia", "Nit", "Clo")))
#group and plot graph
dev_plot <- dev_dat_t2 %>% 
  #select(-Comp, -Stage) %>%
  group_by(Time, Comp, Stage) %>%
  ggplot(aes(x= Comp, y= Worms, fill= Stage)) +
   facet_wrap(~Time, ncol = 3) +
  geom_bar(stat = "identity") +
  ylab ("% worms")


#dev_plot

``` 

(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.

```{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', fig.asp=1.2, include=TRUE, results="hide", echo=FALSE}
#select values 
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),
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          panel.background = element_blank(), 
          axis.line = element_line(colour = "black"),
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          plot.margin = unit(c(5.5,5.5,12,5.5), "pt"),
          text = element_text(size=12, family="sans"))

sel_dev_plot
```

\newpage

## Discussion 
Investigation of the environmental safety profile of pest controlling compounds is essential to ensure safe use of such 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 Nematoda representative *C. elegans* has been investigated.
Limited number of studies 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. 

### Nicotine and neonicotinoids affect locomotion of worms by differential mechanisms
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 on 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 effect no effect on thrashing or body bends. 

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]. BWM is physically attached to the cuticle [@altun2009], therefore prolonged muscle stiffness and hypercontraction lead to shrinkage of the cuticle [@petzold2011] and the reduction in the body size 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 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 a potential molecular targets. 

### Nicotine inhibition of thrashing
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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 to 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 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. 
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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 little material whilst in liquid [@gomez-amaro2015] and so nicotine must cross the cuticle to reach body wall muscle. 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. 

### 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, and/or due to the limited permeability. The increased efficacy of nicotine on wild-type worm motility in 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.

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. 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. 

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*.
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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. 

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\newpage

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------------------------------------------------------
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*.