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UNIVERSITY OF SOUTHAMPTON

FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES

Centre for Biological Sciences

A Comparison of the Effects of Potential Anthelmintics Amidantel, Bay d9216 & Tribendimidine with the Cholinergic Anthelmintic Levamisole

Michelle Joyner

Thesis for the degree of Doctor of Philosophy

October 2012

Michelle Joyner Abstract

UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Biological Sciences Doctor of Philosophy A COMPARISON OF THE EFFECTS OF POTENTIAL ANTHELMINTICS AMIDANTEL, BAY D9216 & TRIBENDIMIDINE WITH THE CHOLINERGIC ANTHELMINTIC LEVAMISOLE By Michelle Joyner

Increasing levels of resistance to currently available anthelmintic drugs has created an urgent need to develop new compounds with the potential to eliminate parasitic worm infections. Amidantel and its derivatives, Bay d9216 and tribendimidine, have previously been reported to act by disrupting transmission at the nematode neuromuscular junction. It is not known whether this activity is mediated by levamisole gated acetylcholine receptors or a novel molecular target. We have used Caenorhabditis elegans as a model organism to investigate the effects of these compounds in wild type worms. A reverse genetic approach was then undertaken, using strains carrying mutations in genes expressing nicotinic acetylcholine receptor (nAChR) subunits or related proteins. We clearly demonstrate that the predominant action of amidantel, Bay d9216 and tribendimidine was an inhibition of locomotion shown on both solid and in liquid media. This activity is in agreement with the previously reported mode of action of these compounds as agonists acting at nAChRs. The level of inhibition was comparable to the widely used anthelmintic, levamisole. Other effects included stimulation of egg laying and reduction in brood size. No effects on the timing of development were observed. A levamisole resistant strain was inhibited by Bay d9216, suggesting a distinct receptor subtype is targeted by this compound. Only a minimal inhibition of locomotion and no reduction in brood size was observed when worms carrying a mutation in the gene expressing the ACR-16 subunit were exposed to Bay d9216. The inhibitory effects of the remaining compounds on this strain were comparable to those of wild type worms. Strains carrying mutations in ACR-12 and ACR-8 subunits showed a reduced susceptibility to Bay d9216. This data suggests that this compound acts by targeting nAChRs containing the ACR-16, ACR-12 and ACR-8 subunits. Susceptibility to Bay d9216 was restored in a strain which was generated to express wild type copies of acr-16 under a body wall muscle specific promoter in an acr-16 mutant background. These data confirmed that the ACR-16 subunit is a key target for the action of Bay d9216. ACR-16 nAChRs have previously been shown to be levamisole resistant. Bay d9216 has also been shown to have activity in economically important parasitic species of worm. Taken together these data provide strong evidence that Bay d9216 has the potential to act as an anthelmintic compound and the molecular target of Bay d9216 is distinct to that of levamisole and therefore has the potential to break resistance.

Michelle Joyner Contents Contents ABSTRACT…...... i Contents……...... ii DECLARATION OF AUTHORSHIP ...... xv Acknowledgements ...... xvi Definitions and abbreviations ...... xvii Chapter 1. Introduction ...... 1 1.1. Helminths ...... 2 1.2. Parasitic worm infections in humans ...... 2 1.3. Parasitic worm infections in agricultural animals ...... 8 1.4. The life cycle of gastrointestinal nematodes ...... 11 1.5. Anthelmintic compounds – an overview ...... 14 1.6. Caenorhabditis elegans ...... 21 1.6.1. The C. elegans life cycle ...... 21 1.6.2. C. elegans as a model organism ...... 23 1.6.3. The evolutionary relationship of C. elegans to other nematode species…...... 26 1.6.4. Physiology of C. elegans ...... 29 1.6.5. Body wall muscle of C. elegans ...... 32 1.6.6. Introduction to neurotransmission in C. elegans ...... 34 1.6.7. Neuronal control of locomotion in C. elegans ...... 41 1.6.8. Neuronal control of feeding behaviour in C. elegans ...... 46 1.6.9. Regulation of brood size in C. elegans ...... 49 1.6.10. Regulation of egg laying behaviour in C. elegans ...... 52 1.6.11. Developmental timing in C. elegans ...... 53 1.6.12. Reverse genetics in C. elegans ...... 54 1.7. Molecular and pharmacological characterisation of nematode nAChRs ...... 56 1.7.1. Molecular characterization of nAChRs ...... 57 1.7.2. nAChRs in C. elegans ...... 62 1.7.2.1. Genes and Nomenclature ...... 62 1.7.2.2. Heterologous expression of nAChRs in Xenopus oocytes .... 65

Michelle Joyner Contents

1.7.2.3. Stoichiometry of nAChRs in C. elegans ...... 67 1.7.2.4. Ancillary proteins associated with nAChRs ...... 72 1.7.2.4.1. RIC-3 – Maturation of nAChRs ...... 72 1.7.2.4.2. UNC-50 - Trafficking of L-AChRs ...... 74 1.7.2.4.3. UNC-74 – Assembly of L-AChRs ...... 76 1.7.3. nAChRs in parasitic nematodes...... 76 1.8. Resistance to anthelmintics ...... 78 1.9. Resistance breaking and novel anthelmintics ...... 81 1.9.1. Amino-acetonitrile Derivatives ...... 81 1.9.2. The acetanilides amidantel, Bay d9216 and tribendimidine ... 84 1.9.2.1. Amidantel and its deacylated derivative, Bay d9216...... 84 1.9.2.2. Tribendimidine ...... 86 1.10. Summary ...... 89 1.11. Project Aims ...... 89 Chapter 2. Materials and Methods ...... 91 2.1. Culturing of Caenorhabditis elegans ...... 92 2.1.1. C. elegans food source ...... 92 2.1.2. Bacterial seeding of plates ...... 93 2.1.3. Freezing/thawing worms ...... 93 2.2. Microscopy ...... 94 2.3. Molecular biology ...... 96 2.3.1. Extraction of Genomic DNA ...... 96 2.3.2. Polymerase Chain Reaction ...... 97 2.3.3. Primer design ...... 97 2.3.4. PCR mixture ...... 98 2.3.6. Plasmids ...... 102 2.3.7. Bacterial Transformation ...... 104 2.3.8. DNA Purification ...... 104 2.3.9. Microinjections ...... 104 2.4. Parasitic worms ...... 107 2.4.1. Ascaris suum ...... 107 2.4.2. Haemonchus contortus...... 108

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Michelle Joyner Contents

2.5. Statistical analysis ...... 108 Chapter 3. Methods for optimal dosing of C. elegans with levamisole, amidantel, Bay d9216 and tribendimidine……………… 109 3.1. Introduction ...... 110 3.2. Methods ...... 112 3.2.1. Preparation of compounds...... 112 3.2.2. Drug Solubility ...... 112 3.3. Assay design ...... 114 3.4. Analysis of locomotion in liquid (thrashing)...... 114 3.4.1. Dosing in liquid for thrashing assays (figure 33)...... 114 3.4.2. Reversibility of compound effects in liquid...... 115 3.5. Analysis of locomotion on solid (agar) medium; body bends .. 115 3.5.1. Dosing for body bends assays; in food (figure 34) ...... 115 3.6. Dosing for body bends assays; in agar (figure 35)...... 117 3.7. Results ...... 119 3.8. Dose-dependence, time-course and reversibility of inhibitory effects with liquid dosing…...... 121 3.9. Estimating the relative potency of compounds ...... 126 3.10. An analysis of the kinetics of compound effects ...... 128 3.11. Time-course for food dosing ...... 130 3.12. Inter Assay variability for food-dosed plates ...... 132 3.13. The effect of the time after food-dosing on the inhibitory action of tribendimidine ...... 134 3.14. Discussion...... 135 3.15. Summary ...... 138 Chapter 4. A comparison of the effects of levamisole, amidantel, Bay d9216 and tribendimidine on C. elegans behaviours and development…...... 141 4.1. Introduction ...... 142 4.2. Methods ...... 143 4.2.1. Pharyngeal pumping ...... 143 4.2.2. Reproduction and survival; Number of progeny per worm .... 144

Michelle Joyner Contents

4.2.3. Egg laying behaviour C. elegans ...... 146 4.2.4. Developmental assays; Timing of development ...... 148 4.3. Results ...... 148 4.3.2. Reproduction ...... 151 4.3.3. Egg laying ...... 153 4.3.4. Development ...... 154 4.4. Discussion...... 161 4.5. Summary ...... 165 Chapter 5. A Reverse genetic screen to identify C. elegans strains with altered susceptibility to amidantel, Bay d9216, tribendimidine or levamisole…...... 167 5.1. Introduction ...... 168 5.2. Methods ...... 173 5.2.1. Thrashing assays of levamisole resistant mutants...... 173 5.3. Results ...... 173 5.3.1. Thrashing assays on levamisole resistant strains ...... 173 5.3.2. Screen of non-levamisole resistant mutants...... 188 5.3.3. Strains with no reported resistance to current anthelmintics……...... 190 5.4. Discussion...... 209 5.5. Summary ...... 211 Chapter 6. The selective interaction of Bay d9216 with ACR-16 .. 213 6.1. Introduction ...... 214 6.2. Methods ...... 214 6.3. Results ...... 214 6.4. Discussion ...... 223 6.5. Summary ...... 224 Chapter 7. The role of nematode body wall muscle N-AChRs in the mode of action of Bay d9216 ...... 227 7.2. Methods ...... 228 7.3. Results ...... 234 7.3.1. The effects of Bay d9216 in wild type, acr-16(+), and acr-16(-) C. elegans...... 234 v

Michelle Joyner Contents

7.3.2. The effects of Bay d9216 in A. suum ...... 236 7.3.3. The effects of Bay d9216 in H. contortus ...... 239 7.4. Discussion ...... 241 7.5. Summary ...... 242 References…...... 257

Michelle Joyner Contents List of figures

Chapter 1 - Introduction Figure 1: Major Human nematode infections, prevalence and distribution……………………………………………………………………...... 6 Figure 2: Major Human platyhelminth infections, prevalence and distribution……………………………………………………………………...... 7 Figure 3: Generalised nematode lifecycle…………………….…….………..13 Figure 4: The chemical structure of piperazine…………………………....15 Figure 5: The chemical structures of levamisole and pyrantel ….....…16 Figure 6: The chemical structure of dihydroavermectin………………….18 Figure 7: The chemical structure of emodepside (PF1022-221)…….…20 Figure 8: The life cycle of C. elegans………………………………….…..….22 Figure 9: The division of the phylum nematoda into clades I-V…...…..27 Figure 10: Adult hermaphrodite C. elegans………………………….….….30 Figure 11: A general view of the C. elegans nervous system………...... 31 Figure 12: The C. elegans neuromuscular junction…………………...... 32 Figure 13: C. elegans in cross section…………………………………...... 33 Figure 14: Cholinergic transmission in C. elegans…………………………37 Figure 15: GABAergic transmission in C. elegans……………………..…..38 Figure 16: The synchronised excitation and inhibition of contralateral body wall muscles in C. elegans…………………………………………..…..43 Figure 17: The connectivity of motor neurons in C. elegans……..…….45 Figure 18: The C. elegans pharynx……………………………………..…….47 Figure 19: The reproductive tract in C. elegans……………………...... 50 Figure 20: Diagram of an α-subunit of a nAChR…………………..……….61 Figure 21: A dendrogram showing the family of genes encoding ACh gated ion channels……………………………………………………..………...63 Figure 22: The basic structure of nAChRs expressed in C. elegans body wall muscle……………………………………………………………..………….68 Figure 23: Acr-16::GFP expression in body wall muscle and DB motor neurons………………………………………………………………..…………...70 Figure 24: ACh responses in wild type and acr-16(ok789) C. elegans..71 vii

Michelle Joyner Contents

Figure 25: The Predicted structure of RIC-3. ………………………………..73 Figure 26: The chemical structure of monepantel………………………….82 Figure 27: The chemical structure of paraherquamide A…………………83 Figure 28: Structures of amidantel and Bay d9216………………………..84 Figure 29: Structures of tribendimidine and levamisole…………………..87 Chapter 2. Materials and Methods Figure 30: Plasmid map for pPD95.86………………………………………103 Figure 31: Injection pads……………………………………………………....106 Chapter 3. Methods for optimal dosing of C. elegans with levamisole, amidantel, Bay d9216 and tribendimidine Figure 32: An investigation of the solubility of Bayd9216…………….113 Figure 33: Analysis of locomotion in liquid………………………………..115 Figure 34: Analysis of locomotion on ‘food-dosed’ plates…………….117 Figure 35: Analysis of locomotion on ‘agar-dosed’ plates…………….119 Figure 36: A Comparison of the locomotory inhibition by cholinergic compounds of each compound with ‘food-dosed’, ‘agar- dosed’ plates or liquid dosing……………………………………………………………………..121 Figure 37: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM levamisole…………………………………………………………..124 Figure 38: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM tribendimidine……………………………………………………..125 Figure 39: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM Bay d9216…………………………………………………………..126 Figure 40: Dose response curve showing the percentage inhibition of thrashing in worms exposed to 1 – 200 µM tribendimidine, Bay d9216 or levamisole in liquid at steady state inhibition…………………………128 Figure 41: Determination of optimal exposure time on food-dosed plates……………………………………………………………………………....132 Figure 42: No significant inter-assay variability was seen between assays on different days…………………………………………………………………134 Figure 43: Tribendimidine stability…………………………………………135

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Michelle Joyner Contents

Chapter 4. A comparison of the effects of levamisole, amidantel, Bay d9216 and tribendimidine on C. elegans behaviours and development Figure 44: Analysis of brood size…………………………………………….145 Figure 45: Analysis of egg laying effects……………………………………147 Figure 46: Comparison of the effects of tribendimidine, amidantel, Bay d9216 or levamisole on pharyngeal pumping rate………………………149 Figure 47: The effects of 10 – 100 µM Bay d9216 or levamisole on pharyngeal pumping rate………………………………………………………150 Figure 48: The effects of Bay d9216, tribendimidine or levamisole on the number of progeny produced by C. elegans adults………………………152 Figure 49: Effects of exposure to Bay d9216 or levamisole on the egg laying behaviour of C. elegans………………………………………………..153 Figure 50: A comparison of the effect of tribendimidine on the timing of development………………………………………………………………………156 Figure 51: A comparison of the effect of amidantel on the timing of development…………………………………………………………………...... 157 Figure 52: A comparison of the effect of Bay d9216 on the timing of development………………………………………………………………………158 Figure 53: A comparison of the effect of levamisole on the timing of development………………………………………………………………………159 Figure 54: The percentage of worms which reached adult stage when allowed to develop on agar-dosed plates (seeded with OP50)………..160 Figure 55: Potential sites of action causing the reduction in progeny number in wild type worms exposed to tribendimidine, Bay d9216 or levamisole………………………………………………………………………….164 Chapter 5. A Reverse genetic screen to identify C. elegans strains with altered susceptibility to amidantel, Bay d9216, tribendimidine or levamisole. Figure 56: Diagram showing the expression pattern of nicotinic subunits and associated proteins in the nervous system……………………...... 172

Michelle Joyner Contents

Figure 57: The thrashing rate of wild type and levamisole resistant C. elegans after 10 and 60 minutes in drug-free buffer…………………...174 Figure 58: The number of body bends generated by unc-50(e306) on ‘food-dosed’ NGM Plates……………………………………………………….176 Figure 59: A comparison of the number of body bends generated by unc-74(e883) on ‘food-dosed’ NGM Plates…………………………………177 Figure 60: The thrashing rate of unc-38(e264) exposed to tribendimidine, levamisole & Bay d9216……………………………………179 Figure 61: The thrashing rate unc-38(x20) when exposed to tribendimidine, levamisole or Bay d9216…………………………………..180 Figure 62: The inhibition of body bends in N2 or unc-38(x20) worms on ‘agar-dosed’ Plates………………………………………………………………182 Figure 63: The number of body bends generated by unc-29(e193) relative to N2 on ‘food-dosed’ plates……………………………………….184 Figure 64: The effect of Bay d9216 or levamisole on the thrashing rate of lev-8(ok1519)…………………………………………………………………186 Figure 65: The effect of Bay d9216 or levamisole on the thrashing rate of lev-1(e211). ……………………………………………………………………187 Figure 66: The effect of Bay d9216 or levamisole on the thrashing rate of acr-23(ok2804)……………………………………………………………….189 Figure 67: The inhibitory effect of Bay d9216 or levamisole on the thrashing rate of acr-14(ok2804)…………………………………………….192 Figure 68: The effect of Bay d9216 or levamisole on the thrashing rate of acr-8(ok1240)…………………………………………………………………193 Figure 69: The effect of Bay d9216 or levamisole on the thrashing rate of acr-12(ok367)…………………………………………………………………194 Figure 70: The effect of Bay d9216 or levamisole on the thrashing rate of acr-16(ok789)…………………………………………………………………196 Figure 71: Summary of the effect of Bay d9216 on the thrashing rate of wild type, acr-12(ok367), acr-8(ok1240) and acr-16(ok789)………….197

Michelle Joyner Contents

Figure 72: The inhibition of body bend generation in N2 or acr- 8(ok1240) worms on amidantel, tribendimidine, Bay d9216 or levamisole plates…………………………………………………………………199 Figure 73: The inhibition of thrashing in N2 or acr-8(ok1240) worms on amidantel, tribendimidine, Bay d9216 or levamisole……………………200 Figure 74: The inhibition of body bend generation in N2 or acr- 12(ok367) worms on amidantel, tribendimidine, Bay d9216 or levamisole plates………………………………………………………………..202 Figure 75: The inhibition of thrashing in N2 or acr-12(ok367) worms in amidantel, tribendimidine, Bay d9216 or levamisole……………………203 Figure 76: The inhibition of body bend generation by acr-16(ok789) worms on amidantel, tribendimidine, Bay d9216 or levamisole………205 Figure 77: The inhibition of thrashing in N2 or acr-16(ok789) worms in amidantel, tribendimidine, Bay d9216 or levamisole……………………206 Chapter 6. The selective interaction of Bay d9216 with ACR-16. Figure 78: The number of body bends generated by N2 or acr-16(ok789) on ‘agar-dosed’ Plates………………………………………………………….215 Figure 79: A comparison of the effects of Bay d9216 on wild type and acr-16(ok789) on ‘food-dosed’ NGM plates…………………………….…216 Figure 80: The effect of Bay d9216 on the number of progeny produced by wild type and acr-16(ok789) C. elegans adults……………………….217 Figure 81: The inhibition of thrashing in acr-16(ok789) exposed to 10 µM tribendimidine, Bay d9216 or levamisole in liquid………………….219 Figure 82: The effect of monepantel on the thrashing rate of N2, acr- 16(ok789) and acr-23(ok2804) worms. …………………………………….221 Figure 83: A summary of the inhibition of thrashing in wild type, acr- 16(ok789) & acr-23(ok2804). …………………………………………………222 Chapter 7. The role of nematode body wall muscle N-AChRs in the mode of action of Bay d9216. Figure 84: The genomic sequence of acr-16………………………………230 Figure 85: Confirmation of deletion in by PCR on genomic DNA isolated from acr-16(ok789)………………………………………………………...... 231

Michelle Joyner Contents

Figure 86: The inhibitory effect of Bay d9216 on the thrashing rate of acr-16(ok789) carrying a wild type copy of acr-16 under the body wall muscle specific promoter myo-3…………………………………………….235 Figure 87: Contraction of A. suum muscle strips in response to Bay d9216, levamisole and nicotine………………………………………………237 Figure 88: Cumulative contraction of A. suum muscle strips in response to Bay d9216 and levamisole………………………………………………….238 Figure 89: A comparison of the number of motile Haemonchus contortus in buffer supplemented with Bay d9216 or levamisole………………….240 Chapter 8. Conclusion. Figure 90: Wnt signalling pathway regulates N-AChR signalling in C. elegans……………………………………………………………………………..255

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Michelle Joyner Declaration List of tables

Chapter 1 - Introduction Table 1: Describing a selection of mutant C. elegans phenotypes with their abbreviated names alongside…………………………………………...25 Table 2: Showing differences between AChR subtypes in A. suum...... 77 Table 3: Reported resistance to anthelmintics by species with mode of action of compounds in C. elegans……………………………………………80 Chapter 2. - Materials and Methods Table 4: C. elegans strain list…………………………………………………..95 Table 5: PCR reaction mixture……………………………………………...... 99 Table 6: PCR cycle conditions…………………………………………………100 Table 7: Expand long template PCR system (Roche) Elongation times………………………………………………………………………………..101 Table 8: Artificial pseudocoelomic fluid (APF) for maintenance of A. suum……………………………………………………………………………….107 Chapter 3. Methods for optimal dosing of C. elegans with levamisole, amidantel, Bay d9216 and tribendimidine Table 9: Summarising the t on rate at maximum inhibition and the 1/2 IC50 at steady state inhibition for tribendimidine, Bay d9216 and levamisole…………………………………………………………………………130 Chapter 4. A comparison of the effects of levamisole, amidantel, Bay d9216 and tribendimidine on C. elegans behaviours and development Table 10: A summary of the effects of levamisole, Bay d9216, amidantel and tribendimidine on C. elegans……………………………………………166 Chapter 5. A Reverse genetic screen to identify C. elegans strains with altered susceptibility to amidantel, Bay d9216 or tribendimidine. Table 11: List of C. elegans mutant strains tested for altered susceptibility to tribendimidine, Bay d9216 or amidantel………….….171

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Michelle Joyner Declaration

Table 12: Summary table showing the effects of tribendimidine, amidantel, Bay d9216 and levamisole on the levamisole resistant strains tested………………………………………………………………………………207 Table 13: Summary table showing the effects of tribendimidine, amidantel, Bay d9216 and levamisole on the non-levamisole resistant strains tested…………………………………………………………………….208

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Michelle Joyner Declaration DECLARATION OF AUTHORSHIP

I, Michelle Joyner declare that the thesis entitled ‘A comparison of the effects of potential anthelmintics amidantel, Bay d9216 & tribendimidine with the cholinergic anthelmintic levamisole’ and the work presented in the thesis are both my own, and have been generated by me as the result of my own original research. I confirm that:  this work was done wholly or mainly while in candidature for a research degree at this University;

 where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated;

 where I have consulted the published work of others, this is always clearly attributed;

 where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work;

 I have acknowledged all main sources of help;

 where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself;

 none of this work has been published before submission.

Signed: ………………………………………………………………………..

Date:…………………………………………………………………………….

Michelle Joyner Acknowledgments Acknowledgements

I would like to thank my supervisors Professors Lindy Holden-Dye, Vincent O’Connor and Robert Walker for their support and guidance throughout the course of my PhD candidature. Thank you to Professor Achim Harder and Dr Claudia Welz at Bayer AG.

I would like to thank all my colleagues in wormland, past and present. Their expertise and assistance have enabled me to conduct this this research. I would particularly like to thank Dr James Dillon for generating the transgenic strains.

I am grateful to Bayer AG, Germany for providing financial support for this research.

Finally I would like to thank my friend Lydia Hall and my family for their support and encouragement during my PhD.

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Michelle Joyner Abbreviations Definitions and abbreviations

ACh Acetylcholine Ami Amidantel Bay Bay d9216 BSA Bovine serum albumin CGC Caenorhabditis Genetics Consortium DHβE Dihydro-beta-erythroidine DMPP Dimethylphenylpiperazinium DMSO Dimethyl sulphoxide GABA gamma-Aminobutyric acid GABAR GABA receptor HSN Hermaphrodite specific neuron 5-HT 5-Hydroxytryptamine L-AChR Levamisole sensitive, nicotine resistant nAChR Lev Levamisole LGIC Ligand gated ion channel MN Motor neuron MOA Mode of action nAChR Nicotinic acetylcholine receptor N-AChR Nicotine sensitive, levamisole resistant nAChR NGM Nematode growth medium NMJ Neuromuscular junction Tri Tribendimidine

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Michelle Joyner Introduction Chapter 1. Introduction

Michelle Joyner Introduction

1.1. Helminths

Helminths (Greek: Worms) exist as parasitic or free living organisms. Two of the helminth phyla are Nematoda (roundworm) and Platyhelminthes (flatworms). Nematodes include the parasitic hookworms, whipworms and filarial worms, and forms the largest of the 34 phyla of the animal kingdom (Girard et al. 2007). Cestodes and Trematodes are major classes of the platyhelminth phyla (Campos et al. 1998) and consist mainly of parasites. Examples from these groups are the blood flukes (trematodes) and tapeworms (cestodes). In addition there are the Turbellaria (which include planarians) which are mainly free-living.

1.2. Parasitic worm infections in humans

Parasitic intestinal helminth eggs have been found in mummified faeces, and clinical features of helminth infections are referred to in Hippocrates and ancient Egyptian writings as well as the bible. They are even accredited with turning events in China during the Cold War. Acute schistosomiasis afflicted Mao’s troops causing them to abort an attack on Taiwan (formerly known as Formosa) and allowing time for American troops to enter the Straits of Taiwan, known as “The blood-fluke that saved Formosa” (Skelly 2008).

Nematode infections

It is estimated that ~two billion people or one third of the world’s population are infected with parasitic worms (Holden-Dye and Walker 2007), the majority of those infected live on less than £1.50 per day and are found in developing countries such as sub-Saharan Africa, Asia and the Americas (Hotez et al. 2008). The commonest infections are the intestinal helminths, followed by schistosomiasis and lymphatic filariasis (LF). Infections are commonly chronic with sufferers often being host to more than one species of helminth (polyparasitized) due to lack of

Michelle Joyner Introduction medical facilities and geographic location. School children are the most common hosts of intestinal worms and schistosomes, they suffer stunted growth, reduced fitness and impaired cognition and memory as a result, leading overall to an impaired education and so a reduced earning capacity (Hotez et al. 2008). Hookworm and schistosomiasis infections during pregnancy regularly cause premature birth, reduced birth weight and maternal mortality. River blindness, caused by Onchocerca volvulus and transmitted by black fly and mosquitoes, is a leading cause of blindness and skin disease in some deprived areas (Hall and Pearlman 1999). Lymphatic filariasis causes major deformities in limbs and genitals (elephantiasis). Overall the effect of infection with parasitic worms in these already impoverished areas promotes poverty and traps the inhabitants in a downward spiral of destitution and disease. The problem is further exacerbated by the coincidence of malaria and/or HIV/AIDS with helminth infections, leading to additive effects such as anaemia and synergistic effects such as increased susceptibility, progression and transmission (WHO 2004).

Platyhelminth infections

Disease in humans due to infection with the pork tapeworm, Taenia sp. is usually asymptomatic, diagnosed by the presence of protoglotids in the faeces. Disease is more serious when the human becomes the accidental intermediate host of tapeworm by ingesting the eggs, usually due to poor sanitation, causing the disease cysticercosis (Muller 1975). Human schistosomiasis is predominant in poor rural areas, peaking in children aged 8-15 years old, and is strongly linked to water contact patterns. Initial penetration by the cercariae can cause a temporary raised, itchy rash, termed ‘swimmers itch’. The migration of schistomulae can induce a hypersensitive reaction known as acute schistosomiasis, with symptoms of fever, fatigue and shadows on the lung. The establishment of the mature worm in the intestinal veins can cause abdominal symptoms which can be severe. Infection lasts 2-10

Michelle Joyner Introduction weeks and sufferers usually recover spontaneously. In chronic infections the eggs can become trapped in tissues during migration. Lytic enzymes are released by the eggs and these can cause an inflammatory and/or granulomatous reaction which lead to the development of fibroids (Gryseels et al. 2006).

Approaches to control

Due to the fact that human helminthiases are prevalent mainly in developing and poor countries, there is very little investment in drug discovery, around 1% of the global investment into drug research goes into anthelmintics, and the majority of this comes from research into drugs targeted to the agricultural market. Because of this lack of investment drugs available to treat human helminthiases are currently very limited. In the three decades from 1975 1,556 new chemical entities (NCEs) were marketed Of these only 4 were developed to treat human infections with helminths, viz albendazole, oxamniquine, praziquantel and ivermectin (Hotez et al. 2008). These, along with diethylcarbamazine, mebendazole and levamisole represent most of the drugs available to humans for curing parasitic worm infections.

Public-private partnerships have been formed under the guidance of WHO to provide mass drug administration (MDA) of anthelmintics, initiated by a public-private agreement to distribute ivermectin by the African programme for Onchocerciasis Control (APOC), in an attempt to treat river blindness in African populations. Drugs for MDA are either donated or low-cost generic drugs, and partners committed to this scheme have formed the Global Network for neglected tropical disease control (GNNTDC) in collaboration with WHO, to raise awareness, funding and political pressure to control and eliminate the most common neglected tropical diseases. The GNNTDC provides support to international organisations, governments and afflicted communities to advocate and implement control and elimination programmes. One of the aims of this alliance is to provide a drug package which targets the 4

Michelle Joyner Introduction four most common helminthiases in humans, Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Ancylostoma duodenale (WHO 2012). Drugs are given irrespective of whether the recipient is infected or not, this strategy is termed preventative chemotherapy. There is some risk that this type of scheme will encourage the emergence of resistance to drugs as has been widely experienced in the veterinary and agricultural industries. This area of concern will require close monitoring (WHO 2008). Examples of helminths that cause human disease are shown in figures 1 and 2. A data bank was established in 2009 by WHO which collected annual data on the number of children aged 1 – 14 years who have received treatment for soil-transmitted helminthiases. These data have revealed that 112 countries or 900 million school aged children have been identified as requiring preventative or curative treatment for helminth infections, with 50% of these children living in just 5 countries. Of those children identified, only 275.3 million actually received treatment which is equivalent to almost 31%. Of the identified countries with endemic helminth infections, Africa contains the largest proportion. 42 African countries were identified with a total of 290 million children requiring treatment for helminth infections and 87 million or 30% actually receiving treatment. The Americas were identified as having the 2nd largest proportion of endemic helminth infections in 30 countries with >45 million children requiring treatment and almost 15 million receiving treatment which represents almost 33% of the school aged population. The target coverage for this age group is 75% by 2020 (WHO 2012).

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Figure 1: Major Human nematode infections, prevalence and distribution. Nematode species, disease caused, regions of highest prevalence, global prevalence. (Information from Hotez et al. 2008; WHO 2008; WHO 2012).

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Figure 2: Major Human platyhelminth infections, prevalence and distribution. Platyhelminth species, disease caused, regions of highest prevalence, global prevalence. (Information from Hotez et al. 2008; WHO 2008; WHO 2012)

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1.3. Parasitic worm infections in agricultural animals

Parasitic worm infections can cause serious clinical disease, welfare problems and loss of production in agricultural animals. As the farming industry has become more intensive the incidence of parasitic worm infections has increased.

Nematode infections

The four main nematodes which cause concern in English sheep farming are Haemonchus contortus, Nematodirus battus, Teladorsagi (previously known as Ostergia) circumcincta and the Trichostrongylus species. H. contortus affects sheep throughout the year, the remaining three affect sheep seasonally. N. battus usually occurs from March to May in southern regions and May to June in the north. T. circumcincta occurs most commonly in the summer and Trichostrongylus strikes at the end of summer and in early autumn. Lambs are at greatest risk as, with the exception of H. contortus, adult sheep develop immunity to these nematodes (Stubbings 2011).

Haemonchus contortus is the most economically important parasite of small ruminants in tropical and sub-tropical regions (Perry et al. 2002). This parasite feeds on the blood of its host and haemonchosis leads to acute anaemia and can be fatal. Annual costs of treating this parasite are estimated to be over £100 million for Kenya, South Africa and India alone (Peter and Chandrawathani 2005). To survive in pasture this parasite prefers minimum average temperatures of 18ºC and monthly rainfall of 50mm. It is commonly found in ruminants raised in warm and wet countries. Recent evidence has shown its occurrence in cooler climates including northern Europe (Peter and Chandrawathani 2005).

T. circumcincta is the most economically important parasitic nematode of small ruminants in temperate regions (Gossner et al. 2012). Infection

Michelle Joyner Introduction with T. circumcincta causes loss of appetite and can lead to weight loss and death.

Nematodirosis, caused by N. battus, of the trichostrongyles family, causes symptoms including diarrhoea and drastic weight loss often leading to death within days in newly weaned lambs (Winter 2002). This has recently become a larger problem in the UK since the first reported outbreak in 1951, occurring in lambs often as a coinfection with other parasitic worms. Stock not killed by nematodirosis can take months to recover. They are also far more susceptible to coinfection with other pathogens or parasites, which can be fatal (Winter 2002).

Platyhelminth infection

Liver flukes are a common agricultural problem in sheep, cattle and horses in the UK, livestock are also commonly afflicted with ostertagiosis, caused by the parasite Ostertagia ostertagi. This parasite also affects young livestock with similar symptoms as nematodirosis. In 2011 50% of cattle livers and approximately 12% of sheep livers were rejected at the abattoir for food use due to damage by flukes and other worms estimated to cost the food processing industry up to £4 million in the UK alone. For each head of cattle infected with flukes it is estimated to cost £30 due to slow weight gain. In infected sheep a reduction in weight gain of between 10 and 30% is estimated which equates to an annual loss of £26,000 in a 500-ewe flock (Cooper 2012).

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Approaches to control

Some parasites such as H. contortus are not present on all farms so quarantine restrictions can be effective at reducing spread of this parasite. Management of pasture has been identified as an important factor in management of helminth infections. This may involve keeping lambs with little immunity on low-risk pasture or swapping pasture use between sheep and cattle to reduce build-up of worm species.

Farmers are often reliant on the “blanket” use of anthelmintic drugs to maintain health of their stock and production levels. Dosing animals too frequently or using an insufficient dose has led to the development of resistance to many of the currently available anthelmintics. It is now recommended that a few of the healthiest members of the flock are not treated with anthelmintics to maintain a reservoir of susceptible nematodes (Stubbings 2011).

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1.4. The life cycle of gastrointestinal nematodes

Intestinal nematodes have a general lifecycle of 4 larval stages (L1-L4) and an adult stage when the worm is reproductive. The larval period is generally free-living and maturation to the adult form occurs within the final or definitive host. The general lifecycle of a nematode is summarised in figure 3.

H. contortus grows to lengths of up to 3cm. The female is larger than the male and can lay 10,000 eggs per day in the abomasum (fourth and final stomach) of the definitive host, usually sheep. These are then excreted in the faeces. The eggs hatch within the faecal pellet as L1, the larvae then shed their cuticle to become L2, feeding on bacteria in the faeces. The 2nd stage larvae undergo an incomplete moult. Retention of the L2 cuticle provides the larvae with protection and nutrients, but prevents it from feeding on bacteria. The larvae remains ensheathed until it is ingested by the definitive host.

Progression through stages L1-L3 takes ~7 days in warm, humid and moist conditions, but can take up to 5 weeks in hot, dry climates, explaining the prevalence of H. contortus in tropical and sub-tropical climates or regions which have heavy summer rainfall (O'Connor et al. 2006). Moisture washes the larvae from the faeces into pasture. L3s, the infective larval stage, migrate up the grass in the presence of sunlight and moisture, increasing the chance of being eaten. L3s can survive drought and cold due to their partial ensheathment (Faust 1939). When ingested the larvae are stimulated rapidly by the gut environment to undergo exsheathment, completing the 2nd larval moult, this is reported to cause a reaction in the gut mucosa causing any previously resident H. contortus to be expelled (Muller 1975). The larvae then migrate to the abomasum undergoing a third and final moult arriving at their final destination as immature adult worms. The adult Haemonchus head measures approximately 30 μm and consists of small buccal cavity with a slightly projecting pharyngeal lancet. It embeds its head in the 11

Michelle Joyner Introduction mucosa, feeding on the blood of the host. Here they mature, mate and reproduce so completing the cycle (Olsen 1986). Progression through the final stages from infective L3 to mature reproductive adult takes between 21-28 days. This species can enter a phase of hypobiosis, allowing the worm to survive adverse weather conditions which could explain the increasing occurrence of H. contortus in climates as cold as Sweden and Denmark (O'Connor et al. 2006).

Ascaris suum is a large roundworm of pigs which causes ascariasis. It grows up to 40 cm in length. Unlike H. contortus this species is ingested at the L2 stage inside the egg. Larvae migrate through the hepatic system before moulting to the L3 stage. They then migrate to the lungs and enter the alveoli where they are coughed up and swallowed. Following this final migration they moult twice to become adults in the pig’s gut.

The lifecycle of A. suum can also be indirect and involve a paratenic host, such as beetles or earthworms. This is a host which is not required for development of the parasite but is used to maintain its lifecycle. The L2 larvae are ingested by the paratenic host and remain in its tissues until it is eaten by the pig and the larvae can complete its lifecycle.

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Figure 3: Generalised nematode lifecycle.

1 – Eggs are excreted from the host in faecal matter

2 – Eggs hatch & develop through larval stages L1 - L3 in the faecal pellet (1-5 weeks). L1 & L2 feed on faecal bacteria and L3 feed on the incompletely moulted L2 cuticle

3 - Moisture washes the larvae out of the faecal pellet

4 - Sunlight and moisture induces the L3s to migrate to the tips of grass

5 – The host ingests L3 with grass, the nematode completes its L3 moult, migrates and attaches itself to the abomasum where it feeds on the blood of the host and matures, becoming reproductive in 21-28 days (information from Olsen 1986).

(B)

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1.5. Anthelmintic compounds – an overview

The geographical distribution of helminthiases in humans is heavily biased towards under developed countries with little money to invest in drug discovery. Due to lack of available funding all the available drugs for human therapy were first developed for the agricultural and veterinary industry (Wolstenholme, Fairweather et al. 2004).

Despite the fact that helminth infections have afflicted humans and animals for centuries it is only in recent years that efficacious and selective anthelmintic drugs have become available. Prior to modern day drugs treatments for internal parasites were mainly based on metals or medicinal plants. The mode of action of most of these compounds was as an irritant or purgative. Some older remedies are included in the following quote:

‘Anthelmintics or worm medicines mechanically irritate the parasites by their speculi, or dislodge them by removing the mucus of the bowels, as purgatives, or prove noxious to the worms themselves – tin or pewter, or iron filed fine, but not levigated, two or three ounces. Common salt, six to eight ounces. Oil of turpentine, two to three ounces, Savine, one to two ounces, Cowhage, half a dram, Calomel, a scruple, Arsenic, ten Grains. Aloes, till they purge’ (Blane 1826).

The first systematic anthelmintic research at Bayer began around 1925 when the earthworm was used as a model to screen potential compounds. This was replaced by two models in 1926, a rodent model using the nematode, Trichinella spiralis and an avian filarial model. Four further models were developed in 1933, but all of these relied on natural nematode infections. The first experimental infection model was developed over the next six years and was accomplished in 1938 using an S. mansoni/mouse model (Harder 2002).

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The development of robust models marked the beginning of a new era in anthelmintic drug discovery. The majority of anthelmintics act on nematode ligand gated ion channels at the neuromuscular junction. The mode of action of some of the commercially available anthelmintic compounds is discussed below.

Phenothiazine was introduced in 1940 and piperazine in 1954 as agricultural anthelmintics. These were the first anthelmintic compounds with selective toxicity. Piperazine is commonly used for threadworm infections in humans. The structure of piperazine is shown below (figure 4). Mode of action (MOA) studies for this compound were carried out in in A. suum where piperazine acts as a weak GABA-mimetic. This compound causes flaccid paralysis and prevents the attachment of the worm in the gut (Martin 1985).

Figure 4: The chemical structure of piperazine Mol. formula: C H N MW 86. 4 10 2,

Thiabendazole was the first member of the structurally related group of benzimidazoles to be discovered in the 1960s. This was closely followed by the discovery of levamisole, the first member of the imidazothiazole group. These compounds were a major improvement on previously

Michelle Joyner Introduction available drugs as they had far superior broad spectrum activity and potency (McKellar and Jackson 2004).

Levamisole and other members of the imidazothiazole group are agonists which act on a subset of nAChRs (Martin 1997).

Pyrantel is a tetrahydropyramidine and it also acts as an AChR agonist at levamisole sensitive nAChRs. In addition pyrantel also has activity as a cholinesterase inhibitor. The chemical structures of levamisole and pyrantel are shown below for comparison.

A B

Figure 5: The chemical structures of levamisole and pyrantel.

(A) Levamisole; mol. formula C H N S, MW 205, (B) Pyrantel; mol. 11 12 2 formula C H N S, MW 206 11 14 2

During the late 1960s and early 1970s further laboratory models for the investigation of anthelmintic drugs were established, including multiple infection mouse models. Praziquantel was synthesised in 1975 as a result of these polyparasitized models. This drug is active against all cestode and most trematode species including Schistosoma sp. Amidantel was also discovered in 1975 and was found to be active against hookworms and Ascaris in dogs. Further development of this compound was stopped at this stage as dosing over consecutive days was required and other anthelmintics for companion animals were available in single dose formulas (Harder 2002).

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Ivermectin was the next milestone discovery in anthelmintic research. This compound was the first of the macrocyclic lactone group, made available in 1981. This drug had activity against a broad range of nematodes and is active at microgram per kg doses (McKellar and Jackson 2004). Ivermectin also has activity against a variety of endoparasites and was released as a broad spectrum endectocide for cattle in 1997 (Harder 2002).

Ivermectin is a semi-synthetic derivative of avermectin. Members of this group are highly complex molecules, unlike the previously described anthelmintic drugs. Other derivatives include moxidectin, milbemycin oxime, doramectin, selamectin, abamectin and eprinomectin.

Ivermectin is a mixture of dihydroavermectin B1a and dihydroavermectin B1b. The structures of these molecules are shown below (figure 6). When exposed to ivermectin persistent potent paralysis of the C. elegans pharynx and body wall muscle is observed. Ivermectin also has a profound effect in the development of larval forms of C. elegans at nanomolar dosages. Macrocyclic lactones interact with many types of ligand gated ion channel (LGIC) including α7 nAChRs, glycine receptors and chloride channels gated by acetylcholine (AChCl-), GABA, histamine or glutamate (GluCl). It is the GluCl channels that are responsible for the anthelmintic activity of ivermectin (Cully et al. 1994).

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Figure 6: The chemical structure of dihydroavermectin B1a and B1b.

Mol. formula C H O + B1B - CH or B1a - C H . 48 72 14 3 2 5 Ivermectin molecular formula (B1a+B1B) C H O , MW 1736 95 146 28

The diketopiperazines group, derived from penicillin, includes marcfortine A and parherquamide. This group was recognised as having anthelmintic activity in 1990. However, they were not widely used due to toxic side effects. Development of a semi synthetic derivative, 2- desoxoparaherquamide A is currently underway, this derivative may be a more attractive candidate for development as this compound shows higher selectivity and an excellent spectrum of activity (Zinser et al. 2002).

For the last quarter century three distinct classes of anthelmintic, the benzimidazoles, the imidazothiazoles and the macrocyclic lactones have been the main commercially available compounds for the elimination of 18

Michelle Joyner Introduction parasitic worms in veterinary and human medicine. Overuse and overreliance on this limited arsenal has led to widespread resistance to each of these groups in parasite targets in sheep, cattle, horses and humans. This increasing resistance has been the impetus behind increasing research into the mechanisms of drug action and resistance in this area (McKellar and Jackson 2004). Alternative delivery methods have been considered in an attempt to overcome resistance such as combinations of compounds with different modes of action (Harder 2002).

Emodepside, shown in figure 7, is a cyclic depsipeptide derived from the fungus Mycelia sterilia. Emodepside has been under development since the 1990s and is now commercially available but only for companion animals (Harder et al. 2003). Anthelmintic activity of emodepside has been shown in multiple species of nematode, including drug resistant strains (Harder et al. 2003). In C. elegans inhibition of pharyngeal pumping, locomotion and egg laying leading to bagging are observed with emodepside. Latrophilin like receptors mediate the pharyngeal pumping effects (Harder et al. 2003), while inhibitory SLO-1 receptors in body wall muscle mediate the locomotion and egg laying effects. SLO-1 is a Ca2+ activated K+ channel homologous to the human BK channel thought to be very important in the action of neuroactive drugs including ethanol and local anaesthetics (Guest et al. 2007). Exposure of C. elegans eggs throughout the life cycle to emodepside has been found to affect developmental timing with a reduced percentage of adults compared to control worms (Bull et al. 2007).

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Figure 7: The chemical structure of emodepside (PF1022-221)

Mol. formula C H N O , MW 1147. 60 90 6 15

Most recently the amino acetonitrile derivative (AAD) groups have been developed. This group of anthelmintics were discovered in 2008 and monepantel was selected for development and commercial release as a broad spectrum anthelmintic with a good tolerability and toxicity profile (Prichard and Geary 2008).

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1.6. Caenorhabditis elegans

Caenorhabditis elegans has been widely employed as an experimental model for anthelmintic studies due to its molecular and genetic tractability. C. elegans is a free living nematode that grows to ~1mm long. It is found in rotting fruit and vegetation and feeds on bacteria. The basic C. elegans anatomy consists of mouth, pharynx, intestine, gonad, and cuticle. The Bristol N2 strain was selected in 1965 by Sydney Brenner as a model organism to study animal development and behaviour, this strain is referred to as wild type throughout this report.

C. elegans is found worldwide. Population genetic studies have shown that migration has occurred over very large distances, even continents; this could have been facilitated by human movement. Despite the small amount of molecular variation there is significant phenotypic variation between environmentally differentiated strains, such as drug susceptibility, locomotory speed and response to pathogens, signatures of local adaptation have not yet been found (Dolgin et al. 2008).

1.6.1. The C. elegans life cycle

The life cycle of C. elegans is 3.5 days from egg to gravid adult at 20 °C, passing through 4 larval stages L1-L4, the average life-span is 2-3 weeks. Figure 8 summarises the C. elegans lifecycle which is far simpler and shorter than that of parasitic worms, requiring no host. In times of food stress or overcrowding C. elegans can enter a dauer stage at L2, similar to the L4 H. contortus. Dauers become thin and cannot eat as their mouths becomes plugged. They are still active during this stage, but are resistant to stress and ageing. They can remain in this state for ~3 months, when the food/overcrowding stress is lifted the worms re- enter the life cycle at L4 and can go on to live ~2 weeks (Croll 1977).

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Figure 8: The life cycle of C. elegans.

Eggs are laid by the adult hermaphrodite and hatch within 14 hours as L1 larvae. L1s moult to become L2 within ~12 hours, in the presence of food and the absence of overcrowding L2 progress to L3 within ~8 hours then moult a further 8 hours later to become L4. A final moult ~10 hours later produces young adult worms, sperm production followed by egg production occurs at this stage. Mature gravid C. elegans then develop within 8 hours (Image taken from Altun and Hall 2012).

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1.6.2. C. elegans as a model organism

Transparency, relatively few cells of fixed number in the adult, ease of cultivation, cryopreservation, microscopic size and amenability to genetic manipulation and analysis are some of the major advantages provided by this organism in the study of many physiological processes (Felix 2008).

C. elegans is a eutelic organism (the adult has a fixed number of cells), having ~40 cells when laid as an egg, 556 cells when newly hatched increasing to 959 somatic cells in the adult hermaphrodite, with a variable number of germ cells. The lineage of every cell from egg to adult has been mapped using laser ablation techniques to individually remove single cells and determine their destined function (Altun and Hall 2012). These methods have revealed that 131 cells die by apoptosis and organs are derived from cells of several different lineages. It is likely developmental control is largely dependent on cell intrinsic signals, although there has been some evidence of inductive signals where a cell will change its path of differentiation to replace killed cells (Croll 1977). The pattern of development is invariable between worms, meaning that the lab strain N2, isolated in Bristol, can be considered to be genetically identical under standard conditions (Dolgin et al. 2008).

C. elegans chromosomes are arranged in 5 pairs of autosomes (I, II, III, IV, and V) and 1 pair of sex chromosomes (XX). Hermaphrodites have two X chromosomes, males are hemizygous, they have one sex chromosome (XO) which can arise from mating or nondisjunction during meiosis leading to the loss of one of the X chromosomes (Sulston and Brenner 1973).

C. elegans was the first multicellular organism to have its genome sequenced in 1998. It has a genome of ~107bp consisting of ~20,500 protein coding genes – a similar number to humans despite having about 1/30 of the genome size which is slightly larger than predicted by 23

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Sulston and Brenner (Sulston and Brenner 1973). There is little repetitive or non-coding DNA, although it is found in the central clusters of the chromosomes.

Ethyl methane sulphonate (EMS) is an alkylating agent widely used to induce point mutations in C. elegans. Mutations are strongly biased towards GC to AT transitions, although some small deletions and other chromosomal arrangements occur. Exposure to 50 mM EMS will induce approximately 20 function affecting variant alleles (Flibotte et al. 2010). Young adult hermaphrodites are selected for mutagenesis and mutant offspring are isolated according to their abnormal appearance, movement or altered susceptibility to drugs. To remove any unwanted mutations these offspring are backcrossed with wild type worms, the lower the dose of mutagen, the fewer backcrossing cycles are necessary (Riddle DL 1977). Males are used in the lab for outcrossing. Mating is an infrequent event in wild populations. Outcrossing involves the mating of genetically different strains. Typically first a genetically modified hermaphrodite is mated with an N2 male, followed by mating the modified offspring with the parental modified strain. This cycle is repeated until the new genetic background is fixed while excluding unwanted genetic changes (Altun and Hall 2012).

Phenotypes of C. elegans are classified by 3-4 letter abbreviations, often relating to the mutated gene, for example strains carrying mutations in genes expressing acetylcholine receptors are frequently abbreviated to acr-n where n refers to the relevant gene number. Abbreviations may relate to drug resistance, for example ‘lev’ strains are resistant to the effects of levamisole. Or physical traits, for example ‘unc’ strains are uncoordinated in their movement, C. elegans can only move in the dorsoventral plane (except for the head) forward or reverse. Mutant strains also include rollers (rol), dumpy (dpy), twitchers and small (sma), long (lon), blistered (bli) and abnormal. Mutations can also affect the pharynx (eat) and the vulva (muv); some of the common phenotypes

Michelle Joyner Introduction resulting from mutations are listed in table 1. The regular occurrence of double mutants has allowed the mapping of genes before the genome was sequenced. It was noted that mutations tended to be highly clustered (Brenner 1973) and it has since been revealed that ~15% of the genes are grouped into operons of 2-8 genes (Riddle DL 1977).

Description Abbreviation

Blistered bli Cell lineage lin Dauer formation constitutive daf-c Degeneration suppression des Dumpy dpy Levamisole resistant lev Long lon Lysosomal cup Multiple vulval development muv Neuronal degeneration deg Pharyngeal effects eat Resistant to inhibitors of cholinesterase ric Roller rol Small sma Systematic RNAi defective sid Uncoordinated unc

Table 1: Describing a selection of mutant C. elegans phenotypes with their abbreviated names alongside.

Further phenotypes can be found at the wormbase phenotype ontology database.

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1.6.3. The evolutionary relationship of C. elegans to other nematode species

Phylogenetic differences of the nematodes and platyhelminths are based on segmentation, pseudocoelom, digestive tract, and sex (Girard et al. 2007).

The similarities found between C. elegans and other nematodes in morphology and development has led to the assumption that structure organisation and regulation of genes would also be very similar between organisms. The phylogenetic relationship of nematodes, based on evolutionary diversity (Proposed by Blaxter et al. 1998) is shown below (figure 9).

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Figure 9: The division of the phylum nematoda into clades I-V

Division is based on expressed sequence tag (EST) projects undertaken by the Genome Project. (Image from Girard et al. 2007).

The free living nematode C. briggsae diverged from C. elegans approximately 100 million years ago. These nematodes are visually very similar, share the same ecological niche and possess the same number of chromosomes and similar genome sizes. Of a predicted 19,500 protein coding genes in C. briggsae, 800 have no orthologs in C. elegans. Operons are also highly conserved in C. briggsae and there is a

Michelle Joyner Introduction surprising amount of variance between genomes of this species and C. elegans (Stein et al. 2003).

The draft sequence from the clade III parasitic nematode Brugia malayi has revealed differences between the genome of this species and that of C. elegans. Genomic conservation between these species was found, but there was an absence of conservation at the local gene level which may reflect evolutionary differences in lifestyle between these free-living and parasitic organisms (Scott and Ghedin 2009). The AT content is higher in B. malayi (69.5%) than in C. elegans (64.6%) or C. briggsae (62.6%). ~15% of B. malayi genes are arranged into operons, like C. elegans, but with greater intergenic spacing and the involvement of different genes. Exons per gene are fewer in B. malayi than in either C. elegans or C. briggsae and are shorter with longer introns (Scott and Ghedin 2009). B. malayi has fewer genes than C. elegans, this may be explained by lineage-specific expansion, of the orthologous genes 8% were expanded in C. elegans (Ghedin and Anton 2009).

There is little synteny between gene order in C. elegans and B. malayi, meaning the gene order of C. elegans could not be used as a scaffold for ordering the B. malayi genome as was expected, extending the time being taken to sequence this genome. There are also a number of genes found in B. malayi that are not found C. elegans (Ghedin and Anton 2009), some of which are found in Drosophila melanogaster and may be present due to this species also being parasitized by a species of Wolbachia (Scott and Ghedin 2009), a symbiotic bacteria which more commonly parasitizes insects including D. melanogaster (Ghedin and Anton 2009).

The B. malayi genome encodes only 7 putative G-protein coupled receptors gated by biogenic amines, such as serotonin. 44 Putative cys- loop genes have been identified in B. malayi, 21 are nicotinic acetylcholine receptor (nAChR) like and 23 are likely to encode chloride channel subunits, compared to ~50 of each in C. elegans. The B. malayi 28

Michelle Joyner Introduction genome has 36 potassium channel genes. Several are orthologous to C. elegans genes (Scott and Ghedin 2009).

The differences between the genomes of these nematodes, previously thought to be very similar, highlight some of the limitations when using a model organism as a platform for mode of action studies. There is a need to confirm the pharmacology of potential anthelmintics in multiple helminths including parasitic species. The presence of B. malayi genes with orthologs in C. elegans may reveal new anthelmintic targets by revealing essential genes in this parasitic species (Scott and Ghedin 2009).

1.6.4. Physiology of C. elegans

The body of C. elegans is shown in figure 10. It is cylindrically shaped and unsegmented (unlike tapeworms of the platyhelminth family), bilaterally symmetrical and tapered at both ends. It consists of two tubes separated by the fluid filled pseudocoelmate cavity which is under internal hydrostatic pressure under the control of an osmoregulatory system (Altun and Hall 2012). The outer tube consists of a collagenous protective cuticle, cuticle secreting hypodermis, muscles, neurons and an excretory system. The inner tube consists of the pharynx, the intestine and gonad (adults). Hermaphrodites have a uterus, 2 ovaries, oviducts and spermatheca. Males have a gonad, vas deferens and fan- like tail. C. elegans exists predominantly as hermaphrodites. Only ~0.05% population are male (Riddle DL 1977).

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Figure 10: Adult hermaphrodite C. elegans.

A – Left lateral side view of adult hermaphrodite, bar = 0.1mm.

B – Representation of anatomical structures from the same view (Image from Altun and Hall 2012).

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The C. elegans nervous system (shown in figure 11) is organised into central ganglia in the head and tail and at neuromuscular junctions (Croll 1977). The circuitry of the neural network of C. elegans was determined in the late 1980’s. To date this is the only organism to have its neural wiring determined.

Microarray, fluorescence activated cell sorting (FACS), and mRNA tagging techniques have been combined to generate embryonic and larval neural expression profiles, providing a model for the generation of spatially and temporally defined reports of neural development and expression (Von Stetina et al. 2007). The nervous system consists of 302 neurons, almost 1/3 of the total cell number, the majority are found in the head and pharynx. 60 of these are ciliated sensory neurons. In the body neuronal cell bodies lie along the midline, there are scattered neurons and smaller ganglia found at the sides. Processes project from the nerve ring in the head, and travel along the dorsal or ventral nerve cord shown in (Croll 1977).

Figure 11: A general view of the C. elegans nervous system

The ventral cord runs from the nerve ring and contains neuronal cell bodies and process tracts. Ventral cord motor neurons send commissures around either side of the body to the dorsal cord. There are four small tail ganglia: the preanal ganglion, the dorsorectal ganglion, and two lumbar ganglia, one on each side (Altun and Hall 2011).

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1.6.5. Body wall muscle of C. elegans

The neuromuscular junction, formed from muscle projections to the outer processes of the motor neurons, control locomotion in C. elegans (figure 12). Body wall muscle is striated and is arranged longitudinally in quadrants, 2 dorsal (DL & DR) and 2 ventral (VL & VR), each quadrant consists of 24 cells except VL which has 23 cells. Muscle attachments to cuticle and hypodermis are equally spaced along the length of the worm. Some non-striated muscle is found in the pharynx, intestine, vulva and rectum (Altun and Hall 2012). The arrangement of C. elegans muscle is shown in cross section below (figure 13).

Figure 12: The C. elegans neuromuscular junction

Neurons (purple, red) are positioned between the hypodermis (beige) and the hypodermal basal lamina (orange line). Arms from the muscles (green) reach the nerves to receive input at neuromuscular junctions that traverse the basal laminae. Synapses between neurons and muscles and between neurons are made en passant at process swellings depicted by arrowheads (Altun and Hall 2011).

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Figure 13: C. elegans in cross section.

(A) The major structures and muscle quadrants

(B) The muscle arms projecting to the dorsal and ventral nerve cords

VR = Ventral right, VL = Ventral left, DR = Dorsal right, DL = Dorsal left. (Image from Altun and Hall 2012).

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1.6.6. Introduction to neurotransmission in C. elegans

Neurotransmission is the rapid transmission of chemical signals across the synapse, a complex and highly regulated process requiring an array of proteins including enzymes, transporters and receptors.

Acetylcholine

Over a third of the neuronal cells in C. elegans release acetylcholine (ACh). It is the major excitatory neurotransmitter at the NMJ, causing contraction of body wall muscle through nicotinic ACh receptors (Rand 2007). These receptors will be discussed in detail in later chapters. Locomotion is the most important ACh mediated behaviour in C. elegans involving the majority of cholinergic neurons (Rand 2007). The characteristic sinusoidal waves or thrashing movement displayed by C. elegans requires waves of contraction generated by ACh release both at the NMJ and in nerve to nerve transmission (Hallam et al. 2000). The rate of wave generation is regulated by the oscillator circuit which also appears to involve cholinergic transmission (Rand 2007).

Other behaviours mediated by ACh signalling include pharyngeal pumping and egg laying. Pharyngeal pumping is stimulated by the cholinergic pacemaker neuron MC (Avery and Thomas 1997).The major effect of ACh in egg laying behaviour is inhibitory, although stimulatory effects have also been reported (Girard et al. 2007).

Developmental timing is also regulated by ACh. When exposed to a specific ACh receptor agonist larval stage L2s were slowed in their developmental timing but the timing of moulting was not affected so the L2/L3 moult occurred before the L3 cuticle was formed (Ruaud and Bessereau 2006).

ACh is synthesised by choline acetyltransferase (ChAT), encoded by the cha-1 gene. Loaded into synaptic vesicles mediated by the vesicular ACh transporter (VAChT), encoded by the unc-17 gene. The driving force for 34

Michelle Joyner Introduction this transport is the acidification of the vesicle by an ATP-dependent proton pump on the vesicle membrane; ACh is exchanged for protons by the VAChT. Docking, priming and calcium dependent fusion of vesicles is not neurotransmitter dependent. Release of vesicle contents into the synaptic cleft occurs after fusion, ACh rapidly crosses the cleft to bind and activate nicotinic ACh receptors (nAChRs) on the post synaptic surface. ACh action is terminated by acetylcholinesterase (AChE), an enzyme which hydrolyses the neurotransmitter. The resultant choline is recycled back into the pre-synaptic terminal, mediated by the high affinity choline transporter (ChT) (Girard et al. 2007).

GABA

The major inhibitory neurotransmitter at the C. elegans NMJ is γ- aminobutyric acid (GABA). While ACh release causes contraction in body wall muscle, GABA acts through inhibitory receptors to cause relaxation during locomotion and resets posture after turning by opening GABA gated chloride channels (McIntire et al. 1993). GABA also has a stimulatory role through excitatory GABA receptors causing a regular contraction of the enteric muscle during defecation by opening GABA gated sodium channels (McIntire et al. 1993). GABAergic signalling is less widespread compared to cholinergic signalling; just 26 of the 302 C. elegans neurons express GABA. Classed according to function, these are D-type motor neurons (6 DD, 13 VD) which innervate the dorsal and ventral body wall muscles, head muscle motor neurons (4 RME), enteric motor neurons (AVL and DVB) and an interneuron of unknown function (RIS) (Girard et al. 2007).

GABA is synthesised by glutamic acid decarboxylase (GAD), this is encoded by the unc-25 gene. Transport of GABA into the vesicle is mediated by the vesicular GABA transporter (VGAT), encoded by the unc- 47 gene, the product of unc-46 is likely to be an accessory protein involved with the VGAT (Schuske et al. 2007). UNC-30 is a transcription factor required for GAD and VGAT. GABA release from DD and VD motor 35

Michelle Joyner Introduction neurons activates the inhibitory GABA receptor encoded by unc-49 in C. elegans; the resultant influx of chloride ions causes the relaxation of the body muscles. GABA release from the AVL and DVB motor neurons activates the novel excitatory EXP-1 receptor; the influx of sodium ions causes the contraction of the enteric muscles and GABA is cleared from the cleft by the plasma membrane transporter, SNF-11 (Schuske et al. 2004).

Diagrams which summarise cholinergic and GABAergic transmission are shown below (figures 14 and 15).

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Figure 14: Cholinergic transmission in C. elegans.

ACh – Acetylcholine, ChAT - Choline acetyltransferase (encoded by cha- 1), VAChT - Vesicular ACh transporter (encoded by unc-17), AChE - Acetylcholinesterase, ChT - Choline transporter (Image from Girard et al. 2007).

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Figure 15: GABAergic transmission in C. elegans.

GAD - Glutamic acid decarboxylase (encoded by unc-25), VGAT - Vesicular GABA transporter (encoded by the unc-47), UNC-30 - transcription factor required for GAD and VGAT. Inhibitory GABA receptor encoded by unc-49. SNF-11 – GABA transporter (Image from Schuske et al. 2004).

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Glutamate

Glutamate is a major excitatory neurotransmitter in the vertebrate central nervous system, acting through the ionotropic glutamate receptors (iGLuRs). C. elegans expresses two types of glutamate receptor. The iGLuR is responsible for both excitatory and inhibitory functions and the invertebrate specific glutamate gated chloride channels (GluCls) have an inhibitory function (Brockie et al. 2001). iGLuRs are expressed in many of the command interneurons (AVA, AVB, AVD, AVE and PVC), controlling forward and reverse locomotion. This appears to be the major role of iGLuRs, although they also have a minor role in other functions including thermotaxis, noxious stimuli avoidance and control of pharyngeal pumping (Raizen et al. 1995; Brockie and Maricq 2006)

GluCl channels have only been identified in invertebrates including C. elegans, Drosophila, parasitic nematodes Haemonchus contortus and Ascaris suum (Cully et al. 1994; Cully et al. 1996; Jagannathan et al. 1999).

Biogenic amines

The biogenic amines serotonin, octopamine, tyramine and dopamine act as neurotransmitters which cause modulation of behaviour in response to environmental cues in C. elegans. Activity in muscles and neurons has been shown. Behaviours affected in response to these neurotransmitters include pharyngeal pumping, egg laying and locomotion (Chase and Koelle 2007).

Exogenous serotonin stimulates pharyngeal pumping (Avery and Horvitz 1990). In the presence of food serotonin activates G-protein coupled receptors to increase pharyngeal pumping and isthmus peristalsis via the activation of SER-7 serotonin receptors located in the MC or M4 neuron respectively (Song and Avery 2012). Application of exogenous 39

Michelle Joyner Introduction octopamine causes the inhibition of pumping and egg laying (Horvitz et al. 1982). Tyramine is a precursor in the biosynthesis of octopamine. It is also likely to act as a neurotransmitter in C. elegans where it acts to inhibit egg laying, modulates reversals and suppresses head oscillations in response to anterior touch (Alkema et al. 2005).

Dopaminergic signalling is involved in the modulation of C. elegans locomotion in response to food. The response to dopamine enables the worm to locate or return to food by using an area restricted search as a foraging strategy (Hills et al. 2004). Dopamine is also implicated in learning in C. elegans. If the plate is tapped it will reverse or increase its rate of locomotion. On repeated tapping wild type worms rapidly habituate to this and no longer respond in the same way. This habituation is modulated by dopaminergic signalling (Sanyal et al. 2004)

Neuropeptides

Small molecule neurotransmitters are located in small clear vesicles clustered at the synaptic zone, whereas neuropeptides are found in dense core vesicles derived from the trans-Golgi network (de Bono and Maricq 2005). Neuropeptides are short amino acid sequences which modulate synaptic activity; they may also have roles as primary neurotransmitters. To date over 250 neuropeptides have been confirmed and are expressed by 113 genes. 40 of these genes encode insulin like neuropeptides (Pierce et al. 2001), 31 encode FMRFamide (Phe-Met-Arg-Phe-NH ) related peptides (Li et al. 1999) and 42 genes 2 encode non-insulin, non-FMRFamide-related peptides (Nathoo et al. 2001). All are expressed as precursors containing single or multiple copies of the same peptide, or multiple distinct peptides, or any mixture of the above. Precursors are processed following translation, starting in the endoplasmic reticulum and continuing in the Golgi and within the dense core vesicles, to yield active peptides at the nerve terminal where vesicles are diffusely scattered (Li and Kim 2008). Neuropeptide genes are expressed extensively through the nervous system and are found in 40

Michelle Joyner Introduction sensory, motor and interneurons. Expression is not limited to neurons and is also found in gonad, intestinal and vulval tissue. Less is known about the role of neuropeptides in modulating C. elegans behaviours but defects on expression have been related to defects in locomotion, dauer formation, egg laying and social behaviour. Neuropeptides may have a modulatory role in all C. elegans behaviours and in addition are likely to have a hormonal role (Girard et al. 2007; Li and Kim 2008).

1.6.7. Neuronal control of locomotion in C. elegans

C. elegans pattern of movement is highly stereotypical, crawling in sinusoidal waves on solid surfaces or swimming in crescent shaped thrashes in liquid. The two major types of locomotory behaviour in this species consist of roaming, in search of food, and dwelling, remaining on food whilst feeding. Both are tightly controlled by the presence or absence of food and the animal’s environment (Ben Arous et al. 2009). Much of C. elegans’ nervous system and over 5% of its genes are devoted to its highly developed chemosensory system, responding to olfactory and gustatory cues which provide information about food, danger and neighbours. Regulation is primarily under the control of the amphid chemosensory neurons, eleven pairs of chemosensory neurons, each detecting a specific subset of attractants, repellents or pheromones (Girard et al. 2007).

Dwelling is a feeding behaviour with slow movement within a limited area in the presence of food. C. elegans spends about 80% of its time dwelling. At infrequent intervals or when off food foraging behaviour is shown consisting mainly of long forwards locomotion interspersed with brief reversals to change direction accompanied by rapid side-to-side movements of the nose in search of food sources (Girard et al. 2007) . Neuropeptides released from interneurons directly postsynaptic to chemosensory neurons regulate dwelling and roaming behaviour by acting on neuropeptide-Y (NPY)/RFamide like receptors (Cohen et al. 2009). 41

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A complex neuronal network involving fast synaptic transmission regulates locomotion at the C. elegans NMJ (Lewis et al. 1980). One GABA receptor (GABAR) and two nAChRs form the functional NMJ. Muscles on the dorsal and ventral surfaces are controlled by concurrent contraction of muscles one side of the worm via excitatory (cholinergic) innervation and inhibition of the opposite side by inhibitory (GABAergic) inputs (Richmond and Jorgensen 1999). This stimulation and contralateral inhibition produces a characteristic sinusoidal movement on solid media which spreads along the entire length of the body to generate body bends leading to coordinated forward or reverse locomotion (figure 16). In liquid medium simultaneous dorsal/ventral excitation and inhibition causes the worm to bend at the mid-section to form a crescent shape and driving a rapid thrashing motion. A similar mechanism of excitation and synchronised inhibition of RME neurons in the head functions via ACh and GABA respectively, this serves to regulate head deflections during foraging and maintain posture (Chalfie et al. 1985; Girard et al. 2007).

The GABAR subunits are encoded by unc-49. The two subtypes of nAChR are distinguished by their sensitivity to levamisole or nicotine (Richmond and Jorgensen 1999). The putative native levamisole receptor is a heteromer encoded by α subunits unc-38, unc-63 and lev-8, and the non-α subunits unc-29 and lev-1. The presence of unc-38, unc-63 and unc-29 are essential for levamisole sensitivity (Fleming et al. 1997). The ancillary proteins RIC-3, UNC-50 and UNC-74 are also essential for the levamisole response highlighting the complex regulation of nAChR signalling (Boulin et al. 2008). In contrast the receptor which is preferentially sensitive to nicotine is proposed to be a homomer of subunits encoded by acr-16 (Touroutine et al. 2005).

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Figure 16: The synchronised excitation and inhibition of contralateral body wall muscles in C. elegans

Showing the stimulation and concurrent inhibition of contralateral muscles which initiates characteristic body bends and leads to coordinated locomotion (adapted from Girard et al. 2007).

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Forward and reverse locomotion in C. elegans is regulated by a well- defined neuronal circuit (Chalfie et al. 1985). Different classes of motor neurons (MNs) are distributed along the length of the animal and innervate a local region of musculature (figure 17). MN cell bodies are located in the ventral nerve cord. The D type MNs are inhibitory and use GABA for neurotransmission, D type neurons DD and VD synapse to dorsal and ventral nerve cords respectively. A and B type MNs are excitatory, using ACh as a neurotransmitter. Cholinergic neurons synapse to the ventral and dorsal body wall muscles and the GABAergic motor neurons (Chalfie et al. 1985; Haspel et al. 2010).

During forward locomotion one set of excitatory and inhibitory neurons controls movement. These are the excitatory dorsal and ventral B type MNs (DB and VB) and the inhibitory dorsal and ventral D class MNs (DD and VD). During reverse locomotion a second set of MNs (DA, VA and DD, VD) are active (de Bono and Maricq 2005). This neuronal connectivity leads to a specific area of musculature contracting due to excitation by cholinergic MNs e.g. VB whilst the contralateral musculature relaxes due to input from the GABAergic MNs e.g. DD (de Bono and Maricq 2005).

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Figure 17: The connectivity of motor neurons in C. elegans

Triangles represent the 6 major motor neuron classes (1 of each shown, multiple members are found along length of ventral cord). Rectangles represent the interneurons involved in the regulation of forward and reverse locomotion (adapted from de Bono and Maricq 2005).

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1.6.8. Neuronal control of feeding behaviour in C. elegans

Pharyngeal muscle is structurally different to body wall muscle. While body wall muscle is striated and longitudinal, pharyngeal muscle consists of radially arranged sectors with each made up of a single sarcomere. Biochemical differences in myosin are also found between body wall and pharyngeal muscle. At the body wall muscle NMJ muscle arms project to nerve cords whereas in the pharynx neurones run within each muscle sector and most synapses to pharyngeal muscle are made en passant (Albertson and Thomson 1976).

When on their bacterial food source, C. elegans feeds by pharyngeal pumping (Avery and Horvitz 1990). The pharynx consists of a corpus, isthmus and terminal bulb. A pharyngeal pump involves the contraction of the corpus, anterior isthmus and terminal bulb. Due to the radially arranged muscle fibres, contraction pulls the lumen open to a triangular cross-section, while the posterior isthmus remains closed so drawing liquid and suspended bacteria into the open corpus. The bacteria are ground as they pass through the contracted terminal bulb (figure 18). Contraction is followed by almost simultaneous relaxation which causes the corpus to close, and bacterial debris to move down to the intestine while expelling any remaining liquid. This contraction-relaxation cycle leads to a rhythmic pattern of pharyngeal pumping (Albertson and Thomson 1976).

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Figure 18: The C. elegans pharynx

(A) Side view of the pharynx showing the main regions

(B) Cross section of the pharynx showing radially arranged muscle cells which pull the lumen open when contracted to allow entry of bacteria suspended in liquid (Adapted from Avery and You 2012).

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Normal, rapid pumping rhythm is regulated by motor neurons including the cholinergic MC neuron which stimulates contraction via the EAT-2 nAChR at the corpus muscle NMJ. EAT-2 is only found at this NMJ however α-bungarotoxin, a nAChR selective probe, binds throughout the pharyngeal muscle surface. Therefore it is likely there are multiple AChR subtypes expressed throughout the pharynx (McKay et al. 2004). Other nAChRs expressed at the C. elegans pharynx include LEV-8 (also known as ACR-13) (Towers et al. 2005). High doses (10 mM) of the cholinergic anthelmintic levamisole reduce the amplitude of electropharyngeogram recordings (Lockery et al. 2012).

Wild type worms pump approximately 250 times per minute (Raizen et al. 1995). In the absence of functional MC, spontaneous action potentials occur leading to slow and irregular pumping. It is not clear how these spontaneous action potentials occur (Avery and You 2012).

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1.6.9. Regulation of brood size in C. elegans

Under normal conditions C. elegans exists primarily as a self-fertilising hermaphrodite (XX) which produces both sperm and oocytes. Males (XO) occur at low frequency (~0.05%) as the result of loss of an X chromosome during meiosis. During the final larval stage (L4) hermaphrodites produce approximately 300 sperm before switching to production of oocytes in much greater numbers (Ward and Carrel 1979). In the absence of mating, wild type C. elegans hermaphrodites will use all of these sperm to produce ~300 progeny (Singson 2001). Therefore self-progeny brood sizes are limited by the number of sperm available (Ward and Carrel 1979). If a male inseminates the hermaphrodite it will use this sperm preferentially and can then produce ~1000 offspring (Croll 1977).

The reproductive tract is a tube-like structure with a distal to proximal opening (gonad tip to exterior). Hermaphrodites have two gonad arms which terminate at the spermatheca, a convoluted tubule where sperm is stored and fertilisation occurs. A distal constriction separates the spermatheca from the gonad arm and a proximal constriction separates each spermatheca from the common uterus which then opens at the central vulval opening through which eggs are expelled. Gametes differentiate as they move proximally (Singson 2001) The hermaphrodite reproductive tract is diagrammed below (figure 19).

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Figure 19: The reproductive tract in C. elegans

(A) An overview of the hermaphrodite reproductive tract.

(B) The regions of the gonad, showing the spermatheca with distal and proximal constrictions, oviduct and uterus (Adapted from Singson 2001).

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Oocytes enlarge and mature before being expelled from the oviduct approximately every 20 minutes (Ward and Carrel 1979). The distal spermatheca dilates allowing entry of the oocyte. Sperm are necessary to trigger oocyte maturation along with several gamete signalling events, these signalling events are yet to be characterised, but are thought to involve a functional LET-23 EGF-receptor, an IP mediated 3 pathway and major sperm protein required for sperm motility (Singson 2001). Spermiogenesis is stimulated in the hermaphrodite when the first oocyte is ovulated from the oviduct which pushes spermatids into the spermatheca from the proximal gonad arm. Sperm then remain in the spermatheca awaiting the next oocyte. Fertilisation of the oocyte occurs in the spermatheca, each oocyte is fertilised by a single sperm, polyspermy is prevented by an unknown mechanism (Ward and Carrel 1979). Signals for the attraction of sperm into the spermatheca and for the binding of sperm with oocyte are also unknown (Singson 2001).

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1.6.10. Regulation of egg laying behaviour in C. elegans

Egg laying behaviour in C. elegans involves the contraction of eight vulval and eight uterine muscles to expel the fertilized egg from the vulva. Vulval and uterine muscle cells are electrically coupled to each other via gap junctions, (White et al. 1986). Multiple neurotransmitters are implicated in the regulation of egg laying behaviour, including 5-HT and ACh. 5-HT is released by the hermaphrodite specific neurons (HSN) which synapse directly with the egg laying muscles (Kim et al. 2001). The VC motorneurons are six hermaphrodite specific neurons located in the ventral nerve cord. Release of ACh from the VC MNs is thought to stimulate muscle contraction directly through nAChRs on the vulval muscle cells while also inhibiting the HSNs through muscarinic AChRs. Other neurotransmitters, including neuropeptides released from the VC MNs may also be involved in the regulation of egg laying behaviour (Schafer 2005). Levamisole and other cholinergic agonists have been shown to induce egg laying behaviour in C. elegans. Of the eight levamisole resistance genes identified seven of these (unc-29, unc-38, unc-63, unc-74, lev-1, lev-9, and lev-10) are implicated in the stimulation of egg laying behaviour by levamisole. They are not required for the normal regulation of this behaviour in the absence of drug so these receptors are involved in promotion of egg laying but not required for vulval muscle function (Trent et al. 1983). The L-AChR which promotes egg laying behaviour in the presence of levamisole appears to be very similar in composition to that of the body wall muscle L-AChR. For this reason regulatory pathways in egg laying are likely to be similar to the regulation of body wall muscle contraction. An exception to this is LEV- 1, a non-essential subunit in body wall muscle responses to levamisole, which is essential for the stimulatory effects of levamisole in egg laying (Kim et al. 2001).

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1.6.11. Developmental timing in C. elegans

The C. elegans lifecycle consists of an embryonic (egg) stage, four larval stage termed L1, L2, L3 & L4 and adulthood. Each larval stage is separated by a moult of the cuticle. Development is genetically programmed, determined by expression of heterochronic genes (Ruaud and Bessereau 2006). This genetic programme can be modified by environmental cues such as temperature, food availability and overcrowding thus development through all stages can take 2 – 4 days depending on environmental conditions (Tobin and Saito 2012).

A variety of developmental events are regulated by heterochronic genes in C. elegans, including the cell division and differentiation, neuronal wiring and dauer formation. Mutations in the heterochronic genes alter the timing of such stage-specific events relative to other unaffected events such as larval moulting (Rougvie 2005).

Nicotinic neurotransmission has been implicated in important signalling pathways in the regulation of timing of development in C. elegans (Ruaud and Bessereau 2006). Exposure to the nicotinic agonist dimethylphenylpiperazinium (DMPP) during development is lethal at the L2/L3 moult as cell division and differentiation is delayed while the moult is not, leading to exposure of an undeveloped L3 cuticle. This pathway requires the nAChR subunit UNC-63, which is expressed neuronally and at the body wall muscle in levamisole sensitive nAChRs and the neuronal hormone receptor (NHR) DAF-12 (Ruaud and Bessereau 2006).

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1.6.12. Reverse genetics in C. elegans

The genetic tractability of C. elegans makes this animal a suitable model for genetic investigation. Investigators may select and study animals carrying known mutations in order to characterise the function and pharmacology of genes or gene pathways. For example genes involved in drug susceptibility, development, or locomotion have been identified and described in this way.

Reverse genetics is a very useful tool for identifying potential drug targets through altered susceptibility in mutant worms. The effects of silencing genes or gene products using RNA interference (RNAi) in C. elegans has become a powerful tool in identifying molecules involved in nicotinic signalling (Agrawal et al. 2003; Dykxhoorn and Lieberman 2005). An RNAi library with 86% of the genome has now been developed. Introduction of RNAi through feeding is successful whereas neurons tend to resist RNAi but methods are being developed to overcome this such as strains that are hypersensitive to RNAi (Matta et al. 2007).

The proposed targets must then be verified and this can be done by transformation of the mutant so rescuing wild type responses. C. elegans is amenable to DNA transformation by microinjection and this tool has been used for many different reasons including to over express genes, to express tagged proteins or to analyse the structure and or function of proteins, DNA or RNA (Mello et al. 1991).

It is possible to introduce transgenes into C. elegans by microinjection of DNA into the distal arm of the gonad of an adult (L4+1) hermaphrodite. The nuclei of the germ cells located here share a central cytoplasm so DNA injected here is likely to reach many of the progeny. If successful the injected DNA will be expressed as large extrachromosomal arrays which are large, heritable, cytoplasmic structures that become stable after a rapid series of recombination 54

Michelle Joyner Introduction events between the injected DNA molecules. Occasionally transgenes are randomly integrated into the genome. Integration into the chromosome is a rare event and is dependent on injection into the oocyte nuclei, homologous recombination into endogenous loci can occur albeit much less frequently (Berezikov et al. 2004).

It is common practice to inject the gene of interest alongside a transformation marker such as GFP. The marker acts as a positive control for transformation and offspring are selected by expression of the transformation marker. Transformed progeny are selected and plated individually; these 1st generation transformants will be either transients or heritably transformed. Transients express small transgenic molecules but these have failed to become large enough to form stable arrays and become inheritable. Heritably transformed animals express assembled arrays which are transmitted in the germ line. Offspring inherit the transformed phenotype in a non-Mendelian pattern (Mello et al. 1991). Subsequent offspring are then selected depending on expression of transformant phenotype.

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1.7. Molecular and pharmacological characterisation of nematode nAChRs

Cholinergic neurons release ACh which binds and activates nAChRs to mediate chemical neurotransmission. Cholinergic activity has been identified in multiple neuronal and non-neuronal cells (Grando et al. 1993; Zia et al. 1997; Wessler and Kirkpatrick 2008). Two types of AChR have been identified and are differentiated according to their selectivity for nicotine and muscarine. The nicotinic AChRs (nAChRs) are ligand gated ion channels (LGICs) whose main role is to mediate fast chemical synaptic transmission at the neuromuscular junction and in neurons, ganglia and interneurons (Lena and Changeux 1997). The muscarinic AChRs (mAChRs) are G-protein coupled receptors that mediate slow metabolic transmission through second messenger cascades in neurons and other tissues such as heart, smooth muscle and glands (Wessler and Kirkpatrick 2008). In this section the nicotinic subtype of receptor is discussed in detail as this subtype is most pertinent with respect to anthelmintic action.

The first studies of nicotinic signalling in worms started 30 years ago when mutants resistant to levamisole were isolated (Lewis et al. 1980). Identification of receptor subunits followed and more recently genes for receptor processing, maturation and trafficking have been identified (discussed in section 1.7.2.4). Downstream genes involved in nicotinic signalling include ryr-1 (also known as unc-68), a ryanodine receptor which regulates body wall muscle contraction by amplifying calcium signals (Maryon et al. 1996), tpa-1 which is a protein kinase C homolog implicated in long term nicotine adaptation (Sano et al. 1995), lev-9 and lev-10, implicated in receptor clustering at the NMJ (Gendrel et al. 2009) and lev-11, a tropomyosin homolog (Kagawa et al. 1997). Only a small selection of the nAChR subunits and associated proteins expressed in C. elegans have been discussed in this section. The large size of this family of receptor subunits may provide a number of potential anthelmintic

Michelle Joyner Introduction targets but may also provide a mechanism of resistance to anthelmintic action via the down regulation of target subunits.

1.7.1. Molecular characterization of nAChRs

The first neurotransmitter receptors to be identified biochemically and functionally were the nAChRs, isolated was from the electric organ of the electric ray, Torpedo californica. The first subunit was sequenced around ten years later (Changeux 1979). The nAChRs from electric fish have been found to be very similar to those found in all vertebrate neuromuscular junctions. A neurotoxin called α-bungarotoxin, from cobra venom selectively binds the nAChR allowing affinity purification of the receptor. It is thanks to these two organisms that this receptor is well characterized and rigorously studied (Stroud and Finermoore 1985).

There is a wide distribution of neuronal and non-neuronal nAChRs in mammalian tissue, including muscles, ganglia, neuroendocrine cells and peripheral blood leukocytes. Behavioural studies in mammalian nAChRs focus on effects on the central nervous system (CNS) receptors. Nicotine and related agonists do have peripheral effects for example at the NMJ, especially with systemic administration of high doses. Conversely, in C. elegans studies involve peripheral receptor activation as the mode of administration of agonists is via environmental medium (Matta et al. 2007).

In the vertebrate peripheral nervous system (PNS) and in the adult neuromuscular junction (NMJ) one type of nAChR is found and consists of α1 β1δε (ε replaces embryonic γ). CNS neurons contain many different 2 subtypes of nAChR made up of α and β subunits. These include the isoforms α2 – α7 and β2- β4 in mammalian tissue. α9 and α10 are expressed principally in sensory, immune and other tissues (Matta et al. 2007).

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Vertebrate CNS nAChRs are grouped by affinity for nicotine. Low affinity (μM) receptors are homomeric, formed from α7 subunits and are pre or post synaptically located (Resembling the nicotine sensitive nAChR in C. elegans). High affinity (nM) nAChRs include the predominant α4 β2 receptors which contain combinations of 2 α and 3 β subunits (Resembling the levamisole sensitive nAChR in C. elegans). Multiple combinatorial possibilities and pre or post synaptic location leads to regionally diverse neuronal responses and subsequent behavioural responses (Matta et al. 2007).

The blockade of nicotine evoked responses by nicotinic antagonists has been employed to determine whether responses are mediated by activation of nAChRs. Many nAChR antagonists are available with different selectivities including mecamylamine which selectively blocks α2- α6 receptors and readily crosses the blood-brain barrier (BBB). It is generally used in doses ranging from 0.1 to 2.0mg kg-1 in vivo although selectivity may be compromised in doses over 1 mg kg-1. Hexamethonium, administered at 1.0 – 10 mg kg-1 cannot cross the BBB so only acts on peripheral receptors. Dihydro-β-erythroidine (DHβE) is more selective for α4β2 nAChRs, and α-conotoxin MII antagonises nAChRs containing α6 or α3 subunits, these receptors are central to the mesocorticolimbic reward pathway (Matta et al. 2007).

The relationship between nicotine concentration and effects in the brain can result in the desensitisation of nAChRs adding further complexity to studies. Desensitisation without depolarisation of the cell in a slowly rising nicotine concentration can occur in some CNS receptor populations. This desensitisation can add to the complexity of studies of nicotine effects in the CNS. Nicotine stimulated neurotransmitter responses including dopamine (DA) release in the nucleus accumbens and increased noradrenaline secretion in paraventricular nucleus are dependent on sufficient nicotine reaching the brain quickly enough. Inhalation of nicotine via cigarette smoking is the most effective mode

Michelle Joyner Introduction of nicotine delivery, followed by intravenous administration at the jugular near the right atrium. Other modes do not deliver a rapid enough nicotine bolus to brain tissue. Rate and duration of nicotine administration can alter the balance of receptor desensitisation and resensitisation (Matta et al. 2007).

There are two types of mammalian neuronal nAChR that have been of particular interest to drug discovery, the α7 homomer with five potential ACh binding sites, defined by adjacent subunits and the α4β2 heteromer with a maximum of two binding pockets between the α4 and β2subunits (Arneric et al. 2007). These ligand binding differences suggest the α7 homomer would be more sensitive to agonist than the α4β2. The five potential ACh binding sites in α7 nAChRs allows a greater range of sensitivity. At very low concentrations of agonist the receptor can be activated, while desensitisation is promoted by high concentrations (Papke et al. 2007)

There are multiple different subtypes of each nAChR subunit (α2-7 & α9- 10, β2-4) leading to diverse pharmacological responses and properties (Matta et al. 2007). The functional receptor is a transmembrane protein consisting of five subunits (Arneric et al. 2007) of varying composition depending on cellular location and species. The assembled subunits are arranged in the membrane to form a central ion channel. Each subunit is formed from four transmembrane regions called TM1-TM4 (figure 20) TM2 residues line the cation channel (Corringer et al. 2000). The conductivity and selectivity of the central cation channel is due to the presence of negatively charged amino acids such as glutamate and aspartate (Robertson and Martin 2007).

The N-terminal extracellular region of each subunit is involved in ligand binding. This area contains two cysteines (cys) separated by thirteen amino acid residues. A disulphide bond is formed between the two cys residues, so forming a cys-loop, a signature motif of this superfamily of

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LGIC which also includes GABA, 5-HT, and glycine (Gly) receptors (Brejc et al. 2001). The cys-loop is involved in assembly of the subunits, localisation of the receptor and gating of the ion channel. C. elegans has the most diverse family of cys-loop LGIC known in any organism. Some of the major groups of cys-loop LGIC mentioned above are targets of anthelmintic drugs (Williamson et al. 2007).

A second vicinal di-cysteine bridge is characteristic of the α-subunit of a nAChR. Subunits which do not contain the 2nd di-cysteine bridge motif are known as non-α subunits (β, δ, ε, and γ). The ACh binding site consists of several discrete regions within the N terminal domain, the cys-loop located at the N terminal is essential for ACh binding (Corringer et al. 2000; Barik and Wonnacott 2009). The existence of non-α subunits leads to the diverse physiology and pharmacology of this receptor. A diagram of an α-subunit is shown in below (figure 20). Receptors are composed of 2 or more α-subunits and 3 or less non-α subunits. ACh binds A, B and C loops of the α-subunit, referred to as the principal component. The complementary component of the nicotinic binding site is composed of D, E and F loops from the non α-subunits (Corringer et al. 2000; Barik and Wonnacott 2009). Loops have variable composition in their different subunits and this leads to variations in binding affinity, receptors may contain different non α-subunits leading to non- equivalent binding sites (Robertson et al. 2002).

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Figure 20: Diagram of an α-subunit of a nAChR.

These subunits assemble into homomers or heteromers to form the functional acetylcholine receptor.

M1-M4 represents the transmembrane (TM) regions consisting of around 20 amino acids. The first cys-loop after the N terminal is characteristic of all subunit in the cys-loop ligand gated ion channel superfamily to which nAChRs belong; acetylcholine interacts with this segment of the N terminal in a functional receptor. The second di-cysteine bridge is characteristic of α-subunits within the AChR family. TM2 (M2) lines the pore in an assembled receptor.

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1.7.2. nAChRs in C. elegans

1.7.2.1. Genes and Nomenclature

Mammals and birds possess 17 genes encoding putative nAChR subunits, while the largest known family of genes encoding nAChR subunits belongs to C. elegans. At least 29 subunits have been identified and divided into five groups according to homology, ACR-16, DEG-3, ACR-8, UNC-38 and UNC-29. Each group is named after the first subunit characterised, usually acr (acetylcholine receptor) these subunits belong to the cys-loop LGIC superfamily (Jones et al. 2007). Figure 21 shows C. elegans AChR subunits arranged in their homologous groups, lines represent diversity between subunits.

This number of subunit genes provides C. elegans with potentially thousands of different receptor subtypes. This level of heterogeneity may provide varying sensitivities to ACh, differences in desensitisation, calcium permeability, distribution and/or trafficking (Qian et al. 2006).

A further 32 orphan subunits do not possess the cys-loop signature of LGICs, but do show close homology to the other nAChR subunits. These orphan subunits remain largely uncharacterised in relation to function or pharmacology and are named lgc (ligand gated ion channels of the cys-loop LGIC superfamily), it is predicted that further lgcs are yet to be discovered, 206 genes with the LGIC motif have yet to be identified, most show greatest similarity to the chloride gated channels (Jones et al. 2007).

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Figure 21: A dendrogram showing the family of genes encoding ACh gated ion channels.

Ion channels with sequence homology but no signature cys-loop are classed as orphan subunits. Subunits are grouped according to homology (coloured areas), named after the first subunit characterised in each group. The ACh gated chloride channels (grey) show more homology to other anion channels. Avian/mammalian nAChRs and 5HT 3 receptors are included for comparison (Brown et al. 2006).

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Initially advances in the identification of nAChRs in C. elegans could only be made using methods such as genetic investigations of phenotyped strains such as neuronal degeneration or resistance to known cholinergic agonists. These methods revealed the genes encoding α- subunits deg-3 (neuronal degeneration), des-2 (degeneration suppression) and unc-38 (uncoordinated) and the non- α subunit genes unc-29 and lev-1 (levamisole resistance). Three further subunits were cloned using cross-hybridization methods, acr-2 and acr-3 (non- α) and acr-16 (α subunit).

The levamisole sensitive receptor consists of 3 essential subunits; UNC- 29, UNC-38 and UNC-63, and 2 non-essential subunits; LEV-1 and LEV-8 (Culetto et al. 2004; Brown et al. 2006). It is as yet unknown if the stoichiometry of the levamisole sensitive receptor is fixed. All of the levamisole subunits are expressed in C. elegans body wall muscles. UNC-29 and LEV-1 belong to the group of nAChRs that shares the closest homology to vertebrate muscle while UNC-38 and UNC-63 both belong to the group with most similarity to insect nAChRs. LEV-8 is also known as ACR-13 and belongs to the nematode specific ACR-8 group.

Levamisole is selective for the nematode neuromuscular junction despite the presence of mammalian homologues. Differences between nematode gene sequences and the mammalian homologues explain this selectivity. In the nematode UNC-38 subunit there is a glutamine at position 153 of the B loop. In the mammalian homolog there is a glycine at this position. Selectivity is lost when an E153 substitution is engineered into the murine homolog which then becomes activated by levamisole (Bartos et al. 2006).

The ACR-16 like group shows closest homology to the vertebrate α7 – 10 nAChR subunits and includes acr-16 (formally known as ce-21) and acr -14. All members of the ACR -16 like group express α-subunits except for acr -14 (Jones and Sattelle 2004). Of this group, acr -16

Michelle Joyner Introduction shows closest homology to the vertebrate α7 receptor and can form homomeric receptor when expressed in Xenopus laevis oocytes, it has not been shown that this subunit forms homomers in vivo (Touroutine et al. 2005). It is likely that heteromers exist within this group due to the presence of acr-14, the non- α subunit in this group.

DEG-3 and ACR -8 groups are unique to nematodes. The UNC-38 group bears a close resemblance to insect nAChRs. The UNC-29 group shows closest homology to vertebrate muscle and Drosophila (ARD) non- α subunits. Some of the orphan subunits, while sharing homology to the nAChRs, more closely resemble the 5-HT, GABA and Glycine receptors, notably lacking negatively charged amino acid residues within the predicted TM2 region. This region determines ion selectivity so these orphan subunits may form anion channels.

All the subunits possess common characteristics of the nAChR such as an N terminal ligand binding domain and four predicted transmembrane regions, there are unexpected alterations in some of the subunits which may lead to modified ligand affinity and/or ion selectivity. So far there is no explanation why such a simple organism possesses such an extensive nAChR gene pool.

1.7.2.2. Heterologous expression of nAChRs in Xenopus oocytes

Heterologous expression of nAChR subunits in Xenopus oocytes has been used to investigate the molecular mode of action of cholinergic anthelmintics and to compare the pharmacology of homologous receptors from different species.

The nicotine sensitive homomeric nAChRs from C. elegans made up of ACR-16 subunits and the vertebrate α7 homolog have been expressed in Xenopus oocytes to compare the pharmacology of these receptors. Responses to nicotine and ACh were found to be very similar while

Michelle Joyner Introduction differences were revealed in their responses to the cholinergic anthelmintic oxantel. This compound acts as a partial agonist on mammalian α7 nAChRs but no action on ACR -16 nAChRs was observed (Sattelle 2009).

Levamisole acts as a cholinergic agonist at a specific subset of nAChRs in C. elegans termed L-nAChRs. This compound has been used to isolate mutants on the basis of resistance, initially mutations in genes encoding LEV-1, UNC-29 and UNC-38 were found to confer resistance to this compound (Brenner 1973). Later electrophysiological investigation of combinations of subunits that included UNC-38 expressed in Xenopus oocytes showed inward currents in response to levamisole which were reduced in the presence of a nicotinic antagonist. The nAChR genes unc- 63 and acr-13 have since been implicated in levamisole resistance (Culetto et al. 2004; Towers et al. 2005). To date there has been no success in attempts to express the ACR -13 (LEV-8) channel, but UNC-63 co-expresses with LEV-1 and UNC-29 to form a functional channel that is sensitive to levamisole. This type of study has shown there is a distinction between levamisole sensitive (L-AChR) and levamisole insensitive (termed N-AChR) receptors which still respond to other nicotinic ligands (Culetto et al. 2004).

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1.7.2.3. Stoichiometry of nAChRs in C. elegans

The C. elegans NMJ contains at least two cholinergic receptor subtypes which have been well characterised. These are the levamisole sensitive nAChR and the nicotine sensitive nAChR (Touroutine et al. 2005).

The levamisole sensitive heteromer is made up of 2 α and 3 non-α subunits of variable composition which may include UNC-38, UNC-63 and UNC-29. The stoichiometry of the nicotine sensitive nAChR is proposed to be homomeric made up of ACR-16 α subunits encoded by acr-16 (previously known as Ce21). The closest vertebrate homolog of this receptor subunit is the homomer forming α7 with 47% homology. (Raymond et al. 2000; Touroutine et al. 2005).

Fusion of GFP to nAChR subunits to investigate spatiotemporal expression patterns has revealed there is often little distinction between expression in muscle and neuronal tissue in C. elegans, whereas vertebrates appear to have distinct muscle and neuronal nAChR subunits (Fleming et al. 1997) nAChRs expressed in other C. elegans tissue, for example neuronal receptors, have not been characterised as fully. A diagram comparing the nicotine sensitive and levamisole sensitive nAChR is shown in figure 22. Alongside these cartoons, the structure of an nAChR resolved to 4.6 angstrom is shown with detail showing the binding site of levamisole and characteristics of the channel, both receptor subtypes are found in C. elegans body wall muscle.

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Figure 22: The basic structure of nAChRs expressed in C. elegans body wall muscle.

(A) The nicotine sensitive homomeric receptor which is insensitive to levamisole. (B) The heteromeric levamisole sensitive receptor which is insensitive to nicotine (α subunits – blue, non-α subunits – yellow). nAChRs gate channels which are non-selective cation channels.

A high resolution structure of a nAChR is shown in (C) resolved to 4.6 angstrom. The asterisks denote the levamisole binding site at the interface between α and non-α subunits, the upper arrow shows the gate which blocks passage of ions when closed and the lower arrow points to the narrowest part of the channel when open (Adapted from Jones and Sattelle 2004).

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The expression pattern of ACR-16 has been elucidated using a transgenic strain expressing acr-16::GFP (Touroutine et al. 2005). Expression was detected in a subset of neurons, particularly the DB motor neurons in the ventral nerve cord and all body wall muscle (figure 23). Expression was also detected in the embryo (Touroutine et al. 2005).

The cholinergic response in C. elegans body wall muscle has been shown to consist of two components. The nicotine sensitive component is a large, rapidly desensitising excitatory current and the levamisole sensitive component is a smaller, slowly desensitising current. The nicotine sensitive current has been shown to be reduced by ~85% in acr- 16(ok789) deletion mutants while the levamisole sensitive current remains largely unaffected (figure 24). This reduced response to ACh was rescued in transgenic lines expressing ACR-16 under the body wall specific myo-3 promoter. In double mutants carrying mutations in acr- 16 and unc-63 which encodes a subunit essential for the levamisole response all cholinergic response was eliminated (Touroutine et al. 2005).

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Figure 23: Acr-16::GFP expression in body wall muscle and DB motor neurons

(A) Ventral view of mid-body region showing acr-16::GFP expression in body wall muscle and DB motor neurons (arrowheads) in the ventral nerve cord. (B) Combined GFP/DIC image showing acr-16::GFP expression in body wall muscle. Spiral appearance of body wall muscle is due to the presence of the rol-6 transgenic marker.

Bar=10 μm (From Touroutine et al. 2005).

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Figure 24: ACh responses in wild type and acr-16(ok789) C. elegans

The representative current traces of voltage-clamped body wall muscle ACh responses (100-ms pulses of 5 x 10 -4 M ACh or levamisole as indicated) in (A) wild type worms and (B) acr-16(ok789), the reduction in ACh current in this strain is ~85% relative to wild type. (Adapted from Touroutine et al. 2005)

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1.7.2.4. Ancillary proteins associated with nAChRs

Genetic studies have revealed genes which encode proteins associated with AChR assembly, trafficking and maturation. A total of 8 genes have been identified as essential for functional reconstitution of the C. elegans levamisole sensitive nAChR in the heterologous Xenopus system. When the recombinant receptor was expressed omitting any of these genes levamisole currents were reduced by 90%, this reduction was exaggerated when ric-3 was omitted. 3 genes encode proteins which are required for receptor assembly and surface expression, ric-3, unc-50 and unc-74 (Boulin et al. 2008).

1.7.2.4.1. RIC-3 – Maturation of nAChRs ric-3 (Resistant to Inhibitors of Cholinesterase) was the first component of the cellular machinery involved in maturation of nAChRs to be identified. Maturation of the receptor leads to production of fully assembled and functional receptors on the plasma membrane. RIC-3 is an endoplasmic reticulum associated protein, comprised of 2TM domains with both N and C terminals and extensive coil-coil domains in the cytoplasmic side. The coil-coil domains are implicated in protein- protein interactions with other coil-coil domains (Halevi et al. 2002) The predicted structure is shown below (figure 25).

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Figure 25: The Predicted structure of RIC-3.

N and C terminals are located on the cytoplasmic side of the membrane along with coil-coil domains which are involved in protein-protein interactions (Information from Halevi et al. 2002).

Expressed in muscles and neurons and concentrated in cell bodies, RIC- 3 is required for at least 4 distinct nAChRs and is involved in the maturation of AChR and 5-HT subunits (Boulin et al. 2008). In ric-3 3a mutants, immunostaining has shown that the neuronal nAChR subunit DEG-3 accumulates in neuronal cell bodies, confirming RIC-3 is not required for deg-3 translation or transcription and suggesting a defect in folding, assembly, or trafficking of the receptor. DEG-3 assembles with DES-2. When RIC-3 is co expressed in Xenopus oocytes the activity of DEG-3/DES-2 and rat α7 nAChRs is increased (Halevi et al. 2002). RIC-3 is not essential for maturation in Xenopus but increases the efficiency of the maturation process. Little is known about the cellular machinery involved in this process (Boulin et al. 2008).

Michelle Joyner Introduction ric-3 was identified during a screen of C. elegans mutants which suppress a dominant mutation in deg-3 (neuronal degeneration). Mutations in ric-3 were also identified in mutants defective in ACh transmission at NMJs (Halevi et al. 2002). Together these findings suggest RIC-3 is required for the function of both neuronal and muscle nAChRs.

ACR-16 was expressed as a homomer in Xenopus oocytes and the receptor was functional, being sensitive to nicotine but not to levamisole. When co-expressed with RIC-3 the inward currents were increased (Boulin et al. 2008). RIC-3 was shown to be required for cholinergic transmission in whole cell voltage clamp recordings on Xenopus oocytes whereas GABA and glutamate currents were not affected showing there were no general defects in synaptic transmission (Halevi et al. 2002).

1.7.2.4.2. UNC-50 - Trafficking of L-AChRs

UNC-50 is required to traffic the assembled L-AChR to the plasma membrane (Eimer et al. 2007). The 301 residue UNC-50 protein is conserved in structure and sequence, particularly in the cytoplasmic regions, between related Caenorhabditis nematode species. There are also yeast and human orthologs but the gene is not found in bacteria. UNC-50 mutants are levamisole resistant with uncoordinated movement typical of worms missing functional L-AChRs (Boulin et al. 2008).

The mammalian homologue, UncL (L=50 roman numerals), is a Golgi based RNA binding protein known to increase surface expression of the vertebrate α4β2 nAChRs (Eimer et al. 2007). UNC-50 interacts with ARF- GEF (Guanine nucleotide exchange factor for ADP ribosylation factor GTPases) which has been shown using yeast 2-hybrid and immunoprecipitation. Active ARF recruits COP1 and other coat proteins to the endoplasmic reticulum membrane. Both point to conserved function (Boulin et al. 2008). 74

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In the absence of UNC-50 L-AChRs are incorrectly targeted to the lysosome and degraded. In the presence of functional UNC-50, L-AChRs are correctly targeted to the plasma membrane (Eimer et al. 2007).

Wild type worms were engineered to express the C-terminally tagged LEV-1-4xHA L-AChR subunit or the UNC-49-3xmyc GABA subunit. When fluorescently labelled antibodies against either of the epitopes were injected into these worms, a punctate staining pattern of GABARs and L- AChRs similar to that of endogenous receptors was seen. This process was repeated with unc-50 mutants and GABARs were expressed at normal levels but no fluorescent signal from Lev-1-4xHA was seen (Eimer et al. 2007).

Immunofluorescence only detects clustered proteins. To confirm the L- AChRs were not present at the plasma membrane in a more diffuse pattern membrane fractions were analysed by Western blot. No L-AChRs were detected in the unc-50 mutant. These findings suggest UNC-50 is specifically required for surface expression of L-AChRs (Eimer et al. 2007)

When UNC-50 was fused to fluorescent protein tags including mRFP- UNC-50 and expressed in body wall muscle a punctate fluorescent pattern typical of localisation in the Golgi was seen. A similar staining pattern was seen with the Golgi marker α-Mannosidase II. When these images were overlaid a large degree of colocalisation was observed. When repeated with an ER marker no overlap was seen. This demonstrated that UNC-50 is localised to the Golgi system in C. elegans (Eimer et al. 2007). unc-50 does not affect DEG-3/DES-2 nAChRs or ACR-16 containing nAChRs so is not a general factor but is specifically required for L-AChRs (Eimer et al. 2007).

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1.7.2.4.3. UNC-74 – Assembly of L-AChRs

Less is known about the role of unc-74 in receptor assembly. Strains carrying unc-74 mutations have the same level of Levamisole resistance as unc-50 (Lewis et al. 1980). UNC-74 is predicted to encode thioredoxin closely related to the human TMX3 (Haugstetter et al. 2005). This protein is required solely for the expression of L-AChRs in vivo and in heterologous expression using Xenopus oocytes. Using heterologous expression, the absence of unc-74 led to a reduction in excitatory currents of more than 90% (Boulin et al. 2008).

1.7.3. nAChRs in parasitic nematodes

The level of diversity of nAChRs seen in the C. elegans family (clade V) is not reflected in other parasitic nematodes. The main C. elegans nAChR subunit groups targeted by anthelmintic drugs are represented in parasitic genomes such as Brugia malayi, a clade III nematode and Trichinella spiralis, a clade I nematode, but these groups are far smaller so targets homologous to C. elegans channels may not be represented (Williamson et al. 2007).

The best described parasitic worm ion channel is that of Ascaris suum (Robertson and Martin 2007). Subtypes of nAChR have been defined in A. suum by pharmacological analysis of responses to levamisole and related compounds, the subtypes are nicotine sensitive, levamisole sensitive and bephenium sensitive (Qian et al. 2006). No B type of nAChR has been identified in C. elegans (Robertson and Martin 2007). The major differences between these channels are summarised in table 2. Further differences have been found in the clade V parasitic nematode Oesophagostomum dentatum which has a 4th conductance subtype, G 40 (Qian et al. 2006) despite being in the same clade as C. elegans.

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The homolog of unc-38 in Trichostrongylus colubriformis is encoded by tar-1. Tar-1 and the H. contortus unc-38 homolog has been found to be 90% identical to unc-38 in C. elegans (Qian et al. 2006).

Subtype N L B

Relative % 28 58 14 proportion of nAChRs

Selective nicotine, levamisole bephenium agonist oxantel, methyridine

Selective paraherquamide 2-desoxo- antagonist paraherquamide

Conductance G G G 25 35 45 subtype

Mean 23 33 45 conductance ps

Channel 0.6 0.9 1.3 open time ms

Table 2: Showing differences between nAChR subtypes in A. suum.

These subtypes have been resolved from patch clamp studies which identified channels with distinct pharmacological and biophysical properties (Qian et al. 2006) and from functional studies on muscle strips on muscle strips showing differential sensitivity to antagonists (Robertson et al. 2002). 77

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1.8. Resistance to anthelmintics

Resistance in agricultural animals

It is clear that resistance to current anthelmintics is becoming an urgent problem in veterinary medicine. This causes both financial and animal welfare problems in addition to concerns about reduced food production. Drug resistance in Haemonchus contortus is now common in sheep (Rufener et al. 2009). Also known as barber’s pole worm, these gastrointestinal roundworms of the trichostrongyloid family cause severe, often fatal anaemia in livestock. Parasitic nematode infections threaten to financially cripple the Australian sheep market due to widespread multiple anthelmintic resistance which continues to rise. 90% of sheep farms tested harboured parasites resistant to the benzimidazoles, resistance to levamisole was found in at least 80% of these farms. Multiple drug resistance was found in at least 60% of the farms surveyed (McKellar and Jackson 2004). Resistance to the macrocyclic lactones (ivermectin) and closantel is now common (Besier and Love 2003).

Anthelmintic resistance is not restricted to Australia and is a global issue. In Spain almost 64% of sheep flocks harboured drug resistant strains of gastrointestinal nematodes. Levamisole resistance was most common (59%) followed by ivermectin (27%) and albendazole (14%) resistance. Multi drug resistant worms were found in 27% of flocks tested with one strain being resistant to all available anthelmintics (Martinez-Valladares et al. 2012). Over the last decade resistance to the benzimidazoles and macrocyclic lactones in Spanish flocks remained at a constant level but the level of resistance to levamisole had almost doubled from 38% in 2003 to over 61% in 2011 (Martinez-Valladares et al. 2012). In the UK studies have indicated almost 100% resistance to the benzimidazoles and 47% resistance to levamisole in lowland sheep farms. In hill farms 83% benzimidazoles resistance was found and 17%

Michelle Joyner Introduction of flocks were harbouring levamisole resistant worms (Jones et al. 2012).

Anthelmintic resistance in cattle is also of concern. The prevalence of resistant infection with Cooperia oncophora in cattle is rapidly increasing (Njue and Prichard 2004). Surveys of cattle parasitized with Cooperia &/or Ostertagia in Argentina showed 64% of herds harboured resistant worms. 60% of the herds were ivermectin resistant, Cooperia was the predominant species. 32% of the herds carried benzimidazole resistant worms, 28 were parasitized by strains of worm resistant to both ivermectin and the benzimidazoles. No resistance to levamisole was reported in cattle (Suarez and Cristel 2007).

Increased incidence of drug resistant nematodes in horses has been reported as has an increase in the number of infections with drug resistant flukes (Wolstenholme et al. 2004).

These are all examples of parasitic worm infections where resistance to currently available anthelmintics is on the rise. Monepantel has ended the drought in new broad spectrum anthelmintics over the last quarter century, currently monepantel is only licensed for use in sheep a limited number of countries including the UK and Australia (Rufener et al. 2010). Emodepside has also become available but is not broad spectrum and is currently only licensed for use in companion animals due to its cost (Hall ; Prichard and Geary 2008).

Anthelmintic drug sales represent the largest proportion of the animal pharmaceutical market, reaching 28.5% in 2009; this translates to £3.36 billion spent globally in this area in one year (McKellar and Jackson 2004). UK farmers spend over £80 million a year on anthelmintic treatments (Cooper 2012). This highlights the importance of effective anthelmintic treatments being available for the agricultural market.

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Some examples of reported anthelmintic resistance in veterinary medicine are listed by species in table 3. Mode of Action in C. Host Parasite Anthelmintic elegans Interacts with β-tubulin, Benzimidazoles ben-1 in C. elegans. Paralysis of body wall muscle and pharynx via Oesophagostomum Ivermectin Pigs GluClα containing spp. receptors. Spastic paralysis of body wall muscle and Pyrantel increased egg laying. AChR agonist. Interacts with β - Benzimidazoles tubulin, ben-1 in C. elegans. Flaccid paralysis. Phenothiazine Competitive antagonist of AChR. Reversible flaccid Horses Small strongyles paralysis. Partial Piperazine agonist of GABA gated Cl- channels Spastic paralysis of body wall muscle and Pyrantel increased egg laying. AChR agonist. Interacts with β - Benzimidazoles tubulin, ben-1 in C. elegans. Spastic paralysis of body wall muscle and Imidazothiazoles Sheep Trichostrongyles increased egg laying. AChR agonist. Paralysis of body wall Avermectin and muscle and pharynx via ivermectin GluClα containing receptors. Interacts with β-tubulin, Cattle Trichostrongyles Benzimidazoles ben-1 in C. elegans.

Table 3: Reported resistance to anthelmintics by species with mode of action of compounds in C. elegans. 80

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Resistance in human infections

The World Health authority (WHO) lists the benzimidazoles, albendazole and mebendazole, and levamisole and pyrantel as essential medicines for the treatment and control of soil transmitted helminthiasis (STH). In endemic areas large scale, regular, single dose administration of anthelmintic drugs at the currently suggested dosage is recommended. The WHO aims to dose 75% of school age children in endemic areas by 2020.

There is concern that widespread anthelmintic administration will lead to development and spread of drug resistant parasitic nematodes. This is not currently a major problem in humans, but of major concern in the veterinary industry. There has been little review or analysis of these drugs efficacy at currently recommended dosage regimes, there is also little consistency in sampling and recording methods when analysing drug efficacy (Keiser and Utzinger 2008).

There are some reports of isolated resistance in human infections. Mebendazole lacked efficacy against hookworms infections in children in Zanzibar and Vietnam (Keiser and Utzinger 2008). This highlights the need for new anthelmintics with increased efficacy and licensed for human use.

1.9. Resistance breaking and novel anthelmintics

As can be seen from the earlier section on anthelmintics (1.5) nAChRs have provided useful targets for the control of nematode infections. This has been borne out by more recent discoveries which also identify nAChRs as their mode of action.

1.9.1. Amino-acetonitrile Derivatives

Amino-acetonitrile derivatives (AADs) have previously been described as having biocidal activity including insecticidal, fungicidal and 81

Michelle Joyner Introduction antibacterial, nematocidal activity has not been described until recently (Ducray et al. 2008). Activity as a new class of anthelmintics was first reported in 2008 (Kaminsky et al. 2008; Prichard and Geary 2008).

In C. elegans AADs affected locomotion, pharyngeal pumping, growth, viability and moulting. Similar phenotypes were observed with the nicotinic agonist, dimethylphenylpiperazinium (DMPP) but not levamisole (Kaminsky et al. 2008). Comparable results were seen in vivo in single and multi-drug resistant H. contortus strains (Ducray et al. 2008; Malikides et al. 2009).

Monepantel was promoted as a candidate for further development due to its low toxicity and good tolerability apparently shared by most of the AADs (Malikides et al. 2009) and if this can also be demonstrated in humans these may be an addition to the limited number of human anthelmintics (Kaminsky et al. 2008). It has broad spectrum efficacy against susceptible, resistant and multi-drug resistant strains due to its novel mode of action through a nematode specific nAChR subunit, ACR- 23 (Kaminsky et al. 2008; Rufener et al. 2009; Rufener et al. 2010). The structure of monepantel is shown in figure 26.

Figure 26: The chemical structure of monepantel

Mol. formula C H F N O S, MW 473.39 20 13 6 3 2

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Paraherquamide and other members of the oxindole family are active against parasitic nematodes in vitro. Unfortunately both compounds induce toxic side effects in mammals (Shoop et al. 1992). The mechanisms of action of paraherquamide and its derivatives have not been completely characterised. They are members of the oxindole alkaloid family isolated originally from penicillium; paraherquamide was isolated from Penicillium paraherqui (Robertson et al. 2002).

Paraherquamide induces flaccid paralysis in parasitic worms in vitro and blocks nicotinic agonists, acting as a competitive antagonist with some selectivity for the L-AChRs (Robertson et al. 2002). Studies on the mode of action of these compounds were undertaken in A. suum where the actions of levamisole were blocked when muscle was pre exposed to paraherquamide (Zinser et al. 2002). The structure of paraherquamide is shown below.

Figure 27: The chemical structure of paraherquamide A

Mol. formula C H N O , MW 494 28 35 3 5

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1.9.2. The acetanilides amidantel, Bay d9216 and tribendimidine

1.9.2.1. Amidantel and its deacylated derivative, Bay d9216.

Amidantel is in a class of compounds called the p-aminophenylamidines. The chemical name of amidantel is N-4-1-(dimethylamino)-ethylidine- amino-phenyl-2-methoxyacetamide (Bay d8815). Amidantel, the parent compound, and its deacylated derivative, Bay d9216, were developed towards the end of the 1970s as anthelmintic drugs against hookworm when it was shown to be effective in rodents against nematodes, filarial worms and cestodes (Wollweber et al. 1979). It was then found to be active against hookworms and ascarids in dogs when administered either orally or subcutaneously (Thomas 1979). The structures of amidantel and Bay d9216 are shown in figure 28.

Figure 28: Structures of amidantel and Bay d9216 The chemical structures of (A) amidantel (mw 249), and (B) Bay 9216/deacylated amidantel (mw 177).

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In human clinical trials infections with Ancylostoma duodenale and Ascaris lumbricoides were cured with single oral doses of amidantel. This compound did not eliminate Necator americanus or Trichuris trichiura infections (Rim et al. 1980; Xiao et al. 2005). Side effects of amidantel were described as transient and mild (Rim et al. 1980).

Amidantel is rapidly metabolised in the host and was detected in the urine as the deacylated derivative Bay d9216 (Rim et al. 1980). The LD 50 in Mice, rabbits, cats and dogs was in the range of 500 – 1000 mg kg-1 (Wollweber et al. 1979).

The effects of amidantel and Bay d9216 on C. elegans were investigated in whole and cut worms (Tomlinson et al. 1985) where the paralysing effects of Bay d9216 were approximately twice as potent as amidantel in whole worms. In cut worms the potency of Bay d9216 was approximately 4 times that of amidantel. Inhibition of AChE was not found. The contractile effects of Bay d9216 were blocked with the cholinergic antagonist tubocurarine and gallamine but not with atropine which is a muscarinic antagonist. These findings led the investigators to conclude that amidantel and Bay d9216 exert their paralysing effects through their properties as cholinergic agonists at the level of the nematode muscle nAChR (Tomlinson et al. 1985).

No effect on the timing of development was reported in C. elegans exposed to amidantel or Bay d9216. Both compounds had an effect on the timing and rate of egg laying which was delayed by approximately 24 hours and reduced by up to 70% of control worms (Woods et al. 1986).

Intracellular recordings made from cholinergic neurons within the sub oesophageal ganglionic mass of the Helix aspersa snail. ‘H’ cells were inhibited by ACh and ‘D’ cells were excited by ACh. The effects of Levamisole, amidantel and Bay d9216 on these cells were compared to

Michelle Joyner Introduction

ACh to determine whether these compounds had a cholinergic mode of action. The relative order of potency for each cell type was:

H cells – ACh>levamisole>>Bay d9216>amidantel

D cells – ACh>Bay d9216>>amidantel=levamisole

Levamisole also showed a slow, irreversible depolarising effect on H cells which resulted in a loss of cell activity (Hassoni et al. 1988).

The potential of these compounds for resistance breaking has not been thoroughly investigated to date.

1.9.2.2. Tribendimidine

Tribendimidine was first synthesised in 1983 based on structure activity relationships (SAR) of derivatives of amidantel. Tribendimidine has been shown to be efficacious against a broad spectrum of nematodes including Nippostrongylus braziliensis in rats, N. americanus in hamsters and Ancylostoma caninum and Toxocara canis in dogs. (Xiao et al. 2005). It was also effective against platyhelminths including the intestinal fluke Echinostoma caproni (Keiser et al. 2006) and several species of cestodes in chicken (Xiao et al. 2005). Tribendimidine was less effective than current anthelmintics against Enterobius vermicularis and T. trichiura infections (Xiao et al. 2005). Efficacy of tribendimidine in the treatment of Opisthorchis viverrini infections was reduced with increased worm burden (Keiser et al. 2008). The structure of tribendimidine is shown in figure 29 below and the structure of levamisole is also shown for reference.

Michelle Joyner Introduction

Figure 29: Structures of tribendimidine and levamisole The chemical structures of (A) tribendimidine (mw 456) and (B) levamisole (mw 241).

Tribendimidine is reportedly fast in onset of action and causes a loss of definition of body wall and organs in trematodes in vivo (Keiser et al. 2006; Keiser et al. 2008).

No acute toxicity or chronic effects were reported with doses up to 250 mg kg-1 given to dogs over fourteen consecutive days. Chronic toxic effects were reported with doses of 510 mg kg-1 over the same period. These tests indicated that the digestive system is the target for tribendimidine when toxic doses are administered (Xiao et al. 2005).

In human trials mild side effects were reported in a small percentage of the trial patients. Tribendimidine was approved for use in humans in China in April 2004 and is now available on the Chinese drug market (Xiao et al. 2005).

Mode of tribendimidine action

A forward genetic screen was used to generate C. elegans which were resistant to tribendimidine. This has shown that tribendimidine acts on

Michelle Joyner Introduction the same receptors as levamisole. Levamisole resistant C. elegans were found to be also resistant to tribendimidine (Hu et al. 2009).

Metabolism

Pharmacokinetic studies in rats showed tribendimidine was widely distributed to all organs and excretion was mainly through urine (Xiao et al. 2005). Liquid chromatography-mass spectrometric (LC-MS) failed to detect tribendimidine in human plasma. Bay d9216 was detected and identified as a metabolite of tribendimidine (Yuan et al. 2008).

Michelle Joyner Introduction

1.10. Summary

The observation of the potent effects of amidantel, Bay d9216 and tribendimidine against a range of parasitic nematodes and the evidence for good tolerance and low toxicity in mammalian host has stimulated interest in these compounds as potentially resistance breaking anthelmintics.

1.11. Project Aims

Anthelmintic compounds are used to treat parasitic worm infections, overuse and overreliance on the limited number of commercially available compounds of this type has led to the development of resistance in many parasitic worm species. Drug resistant parasitic worms infect many types of agricultural livestock and are an emerging problem in human medicine (Keiser and Utzinger 2010). Amidantel and Bay d9216 have both been previously reported to have activity as agonists at nAChRs in C. elegans and H. aspersa neuronal preparations (Tomlinson et al. 1985; Hassoni et al. 1988). Tribendimidine has been reported to have anthelmintic activity in a range of parasitic worm species (Xiao et al. 2005).

The precise mechanism of action by which of each compound attains anthelmintic activity has yet to be fully elucidated. Tribendimidine has recently been reported to act as a nicotinic agonist at levamisole sensitive receptors using C. elegans (Hu et al. 2009). Reports show that both amidantel and tribendimidine are pro-drugs which are rapidly metabolised to Bay d9216 (Rim et al. 1980; Yuan et al. 2008).

This project has utilised the model organism C. elegans to investigate the effects of these compounds on worm physiology and development and to identify the molecular target(s) of these compounds.

Levamisole is known to act as an agonist at a specific subtype of nAChRs at the nematode NMJ. There is significant resistance to this type 89

Michelle Joyner Materials and methods of drug in the field (Neveu et al. 2010). In this study the mode of action of this compound has been investigated and its effects in C. elegans have been compared to those of amidantel, Bay d9216 and tribendimidine. A key goal of this project is to discern any differences between the mechanisms of action of these compounds and levamisole in order that any resistance breaking properties may be identified. Behavioural analyses in wild type C. elegans in addition to a reverse genetic screen have been used to achieve these objectives.

Michelle Joyner Materials and methods Chapter 2. Materials and Methods

Michelle Joyner Materials and methods

2.1. Culturing of Caenorhabditis elegans

C. elegans were maintained and assayed on 5 cm petri dishes containing 14 ml nematode growth medium (NGM). All strains were maintained at 20 °C using standard culture methods (Sulston and Brenner 1974). Strains used are listed in table 4. All strains were obtained from the Caenorhabditis Genetics Centre (CGC).

Composition of NGM (1L) pre-autoclave: 20 g agar, 3 g NaCl, 2.5 g peptone, 972 ml dH O. Post-autoclave (cooled to ~60°C): Using sterile 2 technique & 1 M sterile solutions, 1 ml MgSO , 1 ml CaCl , 25 ml KH PO 4 2 2 4 (pH 6.0) & 1 ml cholesterol (1 mg/ml in ethanol) were added.

Plates were poured using a Jencons perimatic gp pump. Plates were dried for a minimum of 24 hours before seeding with 50 µl Escherichia coli OP50 culture. OP50 (CGC) was cultured on NGM plates for minimum of 48 hours before use. Plates were stored at room temperature for up to two weeks before use.

2.1.1. C. elegans food source

C. elegans were fed and grown on an E. coli OP50 lawn. OP50 was cultured from glycerol stocks, stored at -80 °C, at least annually and as required. For this a sterile swab was used to streak from the glycerol stock, this was spread over the surface of a 9 cm LB agar plate under sterile conditions and incubated overnight at 37°C. Stock plates were stored at 4 °C for up to two weeks. OP50 stocks were maintained by sub culturing, a single colony was picked from the previous stock plate under sterile conditions, using a 1 µl inoculation loop. This was streaked onto a fresh 9 cm LB agar plate and incubated overnight at 37°C. OP50 plates were stored for up to two week s at 4 °C.

Composition of LB agar (1 L) pre-autoclave: 10 g Bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl, 800 ml dH2O, adjust pH to 7.5 with

Michelle Joyner Materials and methods

NaOH, 15 g agar, microwave until agar is melted and adjust volume to 1L with dH2O.

2.1.2. Bacterial seeding of plates

Using a 1 µl inoculation loop, a single OP50 colony was picked and added to 20 ml LB broth in a 25 ml disposable centrifuge tube. This was repeated twice more to give a total of three individual colonies per tube. The broth was incubated overnight at 37°C, with shaking at 225 rpm. The optical density (OD ) of the bacterial broth was measured using an 600 Eppendorf biophotometer and adjusted to an OD of 0.8. Plates were 600 seeded by pipetting 50 µl OP50 broth onto the middle of the NGM at least 48 hours before use. The seeded plates were stored at room temperature.

Composition of LB broth (500 ml) pre-autoclave: 10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl, 475 ml dH O. 2

2.1.3. Freezing/thawing worms

Worms were washed off a well-populated plate in which OP50 had been exhausted the previous day using 2 ml M9 buffer. Worms were pipetted into a sterile 25 ml disposable centrifuge tube. 2 ml freezing solution was added and mixed well. 1 ml of the resultant mixture was then pipetted into each of 4 sterile cryovials. The cryovials were then incubated at -80°C. To test if freezing was successful one of the vials was removed, thawed rapidly and pipetted around the edge of a seeded 5 cm NGM plate. Plates were incubated at 20°C overnight before checking for surviving worms on the food source. If more than five healthy, reproductive worms survived freezing the rest of the batch was then put into permanent frozen storage at -80°C.

Michelle Joyner Materials and methods

Worms were thawed as described for test thaw. Live worms were picked or chunked onto a fresh plate and incubated at 20°C. Thawed strains were not used until F2 progeny were produced.

Composition of worm freezing solution (500 ml) pre-autoclave: 2.9 g NaCl, 3.4 g KH PO , 150 ml glycerol, 2.8 ml 1 M NaOH, 347.2 ml dH O, 2 4 2 aliquot into 5 x 100ml bottles. Post-autoclave: 0.03 ml sterile 1 M MgSO 4 per 100 ml bottle.

Composition of M9 (500 ml) pre-autoclave: 1.5 g KH PO , 3 g Na HPO , 2 4 2 4 2.5 g NaCl dissolve in 499.5 ml dH O then add 0.5 ml 1 M MgSO . 2 4

2.2. Microscopy

C. elegans were viewed at 30 – 62 x magnification using a Nikon SMZ800 stereoscopic zoom microscope with a Nikon CW10XA/22 binocular eyepiece tube and a Nikon WD70 10X lens.

Michelle Joyner Materials and methods

Gene Allele Genotype Strain Description

None None N2 Wild type (Bristol) acr-16 ok789 acr-16(ok789)V RB918 Homozygous. ~1100 bp deletion Superficially wild type acr-16+ ok789 acr-16(ok789); Rescue strain Ex[p-myo-3::acr- acr-16 expressed in 16+] ; [pmyo-2::gfp] body wall muscle & GFP as a transformation marker in pharyngeal muscle acr-16 ok789 acr-16(ok789); Control strain Ex[pmyo-2::gfp] GFP expression in pharyngeal muscle acr-14 ok115 acr-14(ok1155)II RB1132 Homozygous. 5 ~1000bp deletion acr-8 ok124 acr-8(ok1240)X RB1195 Homozygous. 0 ~1050bp deletion, acr-12 ok367 acr-12(ok367)X VC188 Superficially wild type lev-1 e211 lev-1(e211)IV CB211 Levamisole resistant lev-8 ok151 lev-8(ok1519)X VC1041 Levamisole resistant 9 unc-38 x20 unc-38(x20)I ZZ20 Levamisole resistant acr-23 ok280 acr-23(ok2804)V RB2119 Monepantel resistant 4 700bp deletion unc-50 e306 unc-50(e306)III CB306 Levamisole resistant. Recessive. Kinky unc-74 e883 unc-29(e193)I CB883 Levamisole resistant. Recessive. unc-29 e193 unc-29(e193)I CB193 Levamisole resistant. Recessive unc-38 e264 unc-38(e264)I CB904 Levamisole resistant. Recessive unc-63 b404 unc-63(b404)I DH404 Lev resistant Trichlorfon resistant. Table 4: C. elegans strain list

Michelle Joyner Materials and methods

2.3. Molecular biology

2.3.1. Extraction of Genomic DNA

Genomic DNA was extracted from either populations of worms at various developmental stages or from single worms as stated.

To extract genomic DNA from a mixed population worms were washed off a well-populated 5 cm plate with 1 ml M9 & transferred to a 2 ml Eppendorf. This was cooled at 5 °C for ~5 minutes in order that worms settled into a pellet at the bottom of the Eppendorf. The M9 was then pipetted out to leave the pellet in the Eppendorf to which 100 µl of lysis buffer with proteinase K (lysis mix) was added.

This mixture was then incubated at -80 °C for 15 minutes, followed by 60 °C for 1 hour, vortexing every 15 minutes, these steps cause the worm cells to lyse and the enzymatic breakdown of proteins. The final incubation step is at 95 °C for 15 minutes, this is to denature the proteinase K. 300 µl dH O was then added to the lysed worm mixture 2 and this solution was used for PCR reactions.

To extract genomic DNA from single worms, they were first cleaned by transferring to a non-seeded plate for ~ 2 minutes. A single worm was then picked into 5 μl of worm-lysis buffer/proteinase K mix (100 μg ml-1) in a PCR tube. The same procedure for DNA extraction from a population was then followed. PCR reaction mix was added directly to the lysed worm.

Composition of lysis buffer (pre autoclave): 1 ml Tris HCl 1 M pH 8.3, 5 ml KCl 1 M, 0.25 ml MgCl 1 M, 1 ml NP40 45%, 1 ml Tween 2 45%, 10 2 ml gelatin 10X, add dH O to 100 ml. 2

To 99 µl of lysis buffer add 1µl proteinase K 10mg ml-1 just before use.

Michelle Joyner Materials and methods

2.3.2. Polymerase Chain Reaction

Deletions in genomic DNA were detected using the polymerase chain reaction (PCR). This allowed the confirmation of the genotype of the deletion mutant acr-16(ok789). PCR allows the amplification of minute amounts of genetic material for various purposes including the detection of mutations. Three temperature sensitive steps are involved, denaturation annealing and extension.

Denaturation is the melting of dsDNA (template DNA) into single strands so exposing the annealing sites for complementary primers to bind. A DNA polymerase then acts by extending the fragment from the site of primer binding in a 5’ – 3’ direction so generating two identical strands of dsDNA from the original. The cycle is repeated multiple times causing an exponential increase in the number of copies of template DNA.

2.3.3. Primer design

The optimal length for primers is ~ 20 base pairs (bp), oligomers shorter than this carry an increased risk of non-specific binding, resulting in nonspecific products. Longer primers will require excessively high annealing temperatures resulting in reduced yield.

The denaturing and annealing temperatures are determined by the GC content of the primer. The temperature for denaturation (T ) can be m determined by:

T °C = 2(AT) + 4(GC) m

For annealing the temperature is ~ T - 5°C, primer pairs should have m closely matched annealing temperatures, a difference of >5°C may result in no annealing. Temperature is not the only consideration in primer design, GC content of the primer should be 40-60% of the bases, the presence of G or C at the 3’ end promotes specific binding (GC clamp) as these bases bind more strongly than AT. More than 3 G’s or C’s in the 97

Michelle Joyner Materials and methods last 5 bases at the 3’ end should be avoided as should primers which are likely to form a secondary structure such as hairpins and self or cross dimers. These are very likely to lead to little or no yield because the primer would not be available for annealing to the template and amplification would not occur. Primers with repeats or long runs of a single base carry an increased risk of mis-priming leading to nonspecific products. Generally the maximum acceptable number of repeats or runs is 4 bases.

When designing primers areas of cross homology should also be determined to avoid the amplification of genes other than the gene of interest. This possibility can be circumvented by using a search tool such as BLAST to test for specificity.

2.3.4. PCR mixture

For each primer pair a master mix was made up containing all the reactants except for DNA, this mixture is at least 10% more by volume than required for all of the reactions in total. The reaction mix is shown below (table 5), 46 µl of the master mix was added to each PCR tube with 4 µl DNA. For control tubes the DNA was omitted and ddH O added 2 in its place. Primers (Eurofins MWG) and dNTPs (Roche) were in excess.

Two sets of primer pairs were used in two different reactions, forward 1 (F1) + reverse (R) & forward 2 (F2) + R. F1 & R flank the region of interest, F2 is nested within the area of interest, therefore a PCR with F1 + R would be expected to amplify DNA of 1550 bp from wild type worms and 468 bp in the acr-16(ok789) deletion mutant. The nested primer would amplify DNA only in the wild type worms and not in the deletion mutant as the forward primer sits within the deleted sequence.

Each primer was rehydrated to give a stock concentration of 100 µM (100 pmol µl-1). Stock primers were frozen in small aliquots. The final primer concentration used in PCR was 2 µM. Stock dNTPs were 25 mM;

Michelle Joyner Materials and methods the final concentration used in PCR was 750 µM. Expand long template PCR system (Roche) was used. This contains a mixture of Taq DNA polymerase and Tgo DNA polymerase, both are thermostable, Tgo polymerase also has 3’-5’ exonuclease proof reading activity, this mixture copies DNA three times more accurately than Taq DNA polymerase alone. PCRs were optimised through variation of the number of cycles & the annealing temperature.

3 x Master Mix Volume μl / 50 μl Reagent (Final conc.) volume μl reaction

dNTPs 25 mM (750 µM) 1.5 0.5

DNA - 4

Expand long 5 Units/μl 1.5 0.5

Buffer 1 15 5

F1 or F2 Primer 100 μM (2 µM) 3 1

R Primer 100 μM (2 µM) 3 1

ddH O 114 38 2

Total 138 50

Table 5: PCR reaction mixture.

Buffer 1 (Roche) 17.5 mM MgCl

Michelle Joyner Materials and methods

PCR conditions – table 6 shows the cycling conditions used for PCR. Annealing temperature varied depending on the primers being used.

PCRs were run using an M J research PTC-200 Peltier thermal cycler which was pre-heated to 95°C. Samples were placed into the warmed PCR machine in lidded PCR tubes for the required time.

Step Temperature Duration Cycles

Initial 95 oC 2 minutes 1 X denaturation

Denaturation 94 oC 30 seconds

Annealing

Ext primers 58 oC 45 seconds 35 X

Int primers 60 oC

Elongation 68 oC 2 minute

Final elongation 68 oC 7 minutes 1 X

Table 6: PCR cycle conditions. Elongation time is determined by the polymerase, the maximum fragment size expected was 1550 bp (~1.5Kb) 2 minute elongation time was decided upon using the table below as a guide.

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PCR fragment (Kb) 15 20 25 30 35 40 45

Elongation time 11 14 17 20 23 27 30 (min)

Table 7: Expand long template PCR system (Roche) Elongation times. The number of cycles depends on the amount of template DNA used, an increase in the number of cycles may increase yield, but may also increase the error rate.

2.3.5. Gel electrophoresis

1.5% agarose gels were prepared in TBE and allowed to set in a casting tray. 5 µl of 6 x loading dye (Fermentas) was added to each 25 µl sample (final 1 x loading dye). Gels were run with a 1 kb ladder (Invitrogen) at 70 - 80 V in TBE. DNA was visualised using a G:Box Syngene transilluminator, images were captured digitally.

TBE: 45 mM Tris-borate, 1mM EDTA (Fisher)

Agarose gel: 0.5 g agarose (Melford), 50 ml TBE microwaved until agarose fully dissolved. 5 µl GelRed (Biotium) was then added with swirling.

6 X loading buffer: 6% bromophenol Blue 300 μl, Glycerol 30%, H O 70%. 2

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2.3.6. Plasmids

Plasmids are small circular DNAs expressed by bacteria. Their natural function is to transfer genetic information essential for survival of bacterial colonies. In the laboratory they are used to amplify a gene of interest. Plasmids contain a selectable marker such as a gene encoding antibiotic resistance, an origin of replication and a multiple cloning site, containing many restriction enzyme sites which are used to insert the gene of interest.

The pmyo-3::acr-16 cDNA construct pDM867 (Francis et al. 2005) was generated by amplification of acr-16 cDNA from C. elegans and cloning into pPD95.86 (Fire labs, Addgene plasmid 1499). pPD95.86 (figure 30) encodes a gene for ampicillin resistance (AmpR) for selection and the C. elegans promoter (p) for myo-3, a body wall muscle specific promoter.

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Figure 30: Plasmid map for pPD95.86 From Fire Lab, Addgene plasmid 1499

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Michelle Joyner Materials and methods

2.3.7. Bacterial Transformation

Transformation was carried out by Masters Student: Miss Vicky Caves.

LB broth and agar was supplemented with the antibiotic ampicillin to a final concentration of 50µg/ml for selection. The construct was inserted into E. coli DH5α cells (transformation) by incubating 50 µl DH5α cells with the construct on ice for 30 minutes, heat shocked at 42°C for 45 – 60 seconds before incubating again on ice. 900 µl selective LB broth was added before incubating at 37°C with shaking at 150 rpm for 45 minutes. Cells were then plated out on selective LB agar plates and incubated at 37°C overnight.

2.3.8. DNA Purification

DNA was extracted from transformant bacteria using the QIAGEN Plasmid miniprep or maxiprep kits (Qiagen).

Single colonies were picked from freshly streaked selection plates and used to inoculate selective LB broth and incubated overnight at 37°C, with shaking (150 rpm) to produce a starter culture used for purification of DNA. The bacterial cells were pelleted by centrifugation at 10,000 rpm, 4°C. The pellet was re-suspended in buffer and DNA extracted.

2.3.9. Microinjections

Microinjection was performed by Dr James Dillon

It is possible to introduce transgenes into C. elegans by microinjection of DNA into the distal arm of the gonad of an adult (L4+1) hermaphrodite. The nuclei of the germ cells located here share a central cytoplasm so DNA injected here is likely to reach many of the progeny. If successful the injected DNA will be expressed as large extrachromosomal arrays in the offspring of the injected parent, occasionally transgenes are randomly integrated into the genome. It is

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Michelle Joyner Materials and methods common practise to inject the gene of interest alongside a transformation marker such as GFP. The marker acts as a positive control for transformation and offspring are selected by expression of the transformation marker. acr-16(ok789) is a mutant C. elegans strain which expresses a non- functional copy of the nicotinic acetylcholine receptor (nAChR) subunit ACR-16. This strain was injected with the Pmyo-3::acr-16 cDNA construct (pDM867). The coinjection marker used was Pmyo-2::gfp (pPD118.33) (Fire labs), gfp expressed under the control of a (myo-2) pharyngeal muscle specific promoter

DNA solutions were made up in ddH O at concentrations of 40 ng µl-1 for 2 pDM867 & 30 ng µl-1 for pPD118.33. The injection mix and all reactants were kept on ice. Before use the mixture was spun three times at 13000 rpm.

Microinjection needles were pulled from 1 mm diameter aluminosilicate glass capillaries using the P-2000 Sutter Instrument electrode puller. These were stored in an airtight box until use.

The needle was placed in the Eppendorf containing injection mix which is drawn up by capillary action over a few minutes before mounting on a micro-manipulator (Eppendorf TransferMan NK2). The needle tip was broken by tapping it against the broken edge of a coverslip immersed in oil. The position of the manipulator holding the needle was then set for injection.

2% agarose pads were used to hold worms in position. 2% agarose in dH O was made up and drop of this was pipetted onto a glass 2 microscope slide and flattened by placing the agarose slide between 2 slides covered in masking tape injection pads were dried at 60°C for 1 hour before use or storage in an airtight container at room temperature. Before use stored pads were heated briefly on a heat block at ~65°C.

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Agarose Flatten with 4th slide

Injection pad

Taped slides

Figure 31: Injection pads To put the worm on the slide oil was dotted onto the slide beside the gel pad before picking a worm into the oil. The worm was then transferred from oil to the pad. A drop of oil was immediately place over the worm to stop it from drying out. The slide was moved to the injecting microscope and positioned before injecting DNA at 40 x magnification. Following injection a drop of M9 buffer was pipetted onto the worm to disperse the oil and release the worm. The worm was allowed to rehydrate and transferred to a new plate. Injected worms were incubated at 20°C. Transformed progeny were selected by pharyngeal expression of GFP.

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2.4. Parasitic worms

2.4.1. Ascaris suum

A. suum were collected from Mutch Meats (Witney, OX29 7GX), from pigs which had been slaughtered that morning. Worms were transported in a thermos flask containing room temperature artificial pseudocoelomic fluid (APF). They were transferred to APF warmed to 37°C within a few hours of collection. A. suum were maintained in APF at 37°C for up to 4 days, APF was refreshed twice daily and any unhealthy worms (based on colour and movement) were disposed of.

Reagent Concentration MW g/l mM

NaCl 67 58.44 3.92

CH COONa.3H O 67 136.08 9.12 3 2 (Sodium acetate)

CaCl .2H O 3 147 0.44 2 2

MgCl 15.7 95.21 1.49 2

KCl 3 74.55 0.22

Trizma® base 5 121.14 0.61 (tris)

Glucose 3 180.16 0.54

pH 7.6 with glacial acetic acid at 37°C added slowly, ~1750 µl/10 l

Table 8: Artificial pseudocoelomic fluid (APF) for maintenance of Ascaris suum

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2.4.2. Haemonchus contortus.

Wild type (SHco) and the multi-drug resistant White River strain (RHco) were provided by The Moredun Research Institute. The White River strain is resistant to ivermectin, benzimidazoles, rafoxanide and closantel (Le Jambre et al. 1995). L3 larvae were stored in H O at 4°C. 2

2.5. Statistical analysis

All data were analysed using GraphPad Prism 5 (GraphPad Software, San Diego, USA). Data are presented as the mean average (+/- SEM). Statistical significance was determined using either Students t-test, one- way or two-way ANOVA as stated with Bonferroni post hoc test.

(P<0.05 = *, P<0.01 = **, P<0.001 = ***).

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Michelle Joyner Results 1 Chapter 3. Methods for optimal dosing of C. elegans with levamisole, amidantel, Bay d9216 and tribendimidine

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Michelle Joyner Results 1 3.1. Introduction

Amidantel, Bay 9216 and tribendimidine are compounds which have been identified as having anthelmintic potential (Wollweber et al. 1979; Xiao et al. 2009). See section 1.2.2 for structures. The effects of these compounds on worm motility are similar to those of levamisole. However it has not been unequivocally resolved whether or not these compounds all share the same mode of action i.e. the L-AChR. This is important information as a distinct mode of action for any of these compounds may confer levamisole-resistance breaking properties which could be of benefit to veterinary medicine. Furthermore, a distinct mode of action may identify new anthelmintic targets against which further new compounds could be screened.

In this thesis C. elegans has been deployed as a model organism in which to investigate the mode of action of these compounds. However, to date there is scant literature from which to design experimental protocols for which the method of application of the acetanilides is optimal. Particularly important considerations are the stability of the compound on agar plates, with or without E. coli, and the ability of the compounds to get access to the internal milieu of the worm, either by ingestion or absorption across the cuticle.

Previous studies comparing the action of amidantel and Bay d9216 on whole and cut C. elegans allows one to make some predictions about whether or not the drugs can penetrate the cuticle (Tomlinson et al. 1985). The concentration required to elicit paralysis in whole worms was three orders of magnitude higher than in the cut worms suggesting the cuticle does present a barrier to the compounds (350 µM to 0.3 µM for amidantel and 180 µM to 0.07 µM for Bay d9216). In the same study 4 µM and 0.15 µM levamisole was required to paralyse whole and cut worms respectively. Tribendimidine is reportedly fast in onset of action in parasitic nematodes (e.g. in vitro N. americanus; (Xiao et al. 2005; 110

Michelle Joyner Results 1 Keiser et al. 2008)) suggesting that drug access is not a problem. In studies on C. elegans tribendimidine has been reported to cause an inhibition of locomotion but a detailed analysis of the time-course and dose-dependency has not yet been provided (Hu et al. 2009).

Therefore, as a first step to underpin subsequent studies in which the effects of the compounds are compared in different behavioural assays (chapter 4) and in different genetic backgrounds (chapter 5), the efficacy of different dosing regimens has been evaluated in a systematic manner.

The thrashing assay was chosen to investigate the dose-dependency, kinetics, reversibility and relative potency for all the compounds. This assay has been widely deployed for studying C. elegans motility (Buckingham and Sattelle 2008; Buckingham and Sattelle 2009) and has the advantage that the behaviour, thrashing, is very reproducible and can be readily assayed at multiple time points across an extended time course. Furthermore, as the worms are fully immersed in the liquid containing the compound one can control in a relatively precise manner the dosage applied. However, some C. elegans strains, including those with mutations in nAChR which will be the subject of later investigations in the project (chapter 5), thrash irregularly or at a very low rate. Thus in order to investigate the actions of these compounds on C. elegans behaviour a different route of dosing must be used. For this reason, the application of the compounds either through addition to food (i.e. added to the E. coli lawn) or dissolved in the agar was also investigated in order to evaluate the optimal protocol.

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Michelle Joyner Results 1 3.2. Methods

3.2.1. Preparation of compounds.

Amidantel & tribendimidine (Bayer) were dissolved in 100% dimethyl- sulphoxide (DMSO) due to low solubility in aqueous or ethanol solutions. Bay d9216 (Bayer) & levamisole (Sigma) were dissolved in 100% ethanol, dH O or buffer as stated. Compounds were used the same day as they 2 were made up. Stock solutions were sonicated, using a Branson 2510 Sonicator, for fifteen minutes before being diluted in serial steps in M9 or NGM to obtain assay solutions ranging from 10-4 – 10-6 M in 0.1% solvent for all assays unless otherwise stated. Preceding each dilution step and before use solutions were vortexed using a Scientific industries Vortex Genie™ 2. Plates and solutions containing compound were stored away from light except when in use.

3.2.2. Drug Solubility

To determine whether Bay d9216 remained in solution or was held in a suspension at assay concentrations a spectrophotometric assay was conducted.

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Figure 32: An investigation of the solubility of Bayd9216. Solutions were made up in triplicate. The absorbance of a 10 µM solution of Bay d9216 in a final concentration of 0.005% DMSO was measured using a Nanodrop ND-1000 spectrophotometer over 220-350 nM. Solutions were then spun, using an Eppendorf 5417c centrifuge, in 1.9 ml Eppendorf tubes at 14000 rpm/20817 relative centrifugal force (rcf) for 30 minutes. 3 x 1 µl samples were taken from the top of each tube and the absorbance measured again. Samples were kept at room temperature (22°C) throughout.

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Michelle Joyner Results 1 3.3. Assay design

All test compound effects were compared to solvent or buffer controls taken on the same day. The effects of the test compounds were also compared throughout to those of levamisole.

3.4. Analysis of locomotion in liquid (thrashing).

To measure swimming locomotion in liquid (thrashing), the number of thrashes generated by individual worms in one minute is counted. A thrash is defined as the movement in which the midsection of the worm bends to create a crescent shape returns to original position then repeats in the opposite direction and returns to the original position.

3.4.1. Dosing in liquid for thrashing assays.

An L4 + 1 day worm was added to 1800 µl M9 supplemented with 0.1% BSA in a 2 cm Petri dish and allowed to settle for 5 minutes before counting the number of thrashes in 1 minute. 200 µl of compound, vehicle or buffer was then added to give a final volume of 2 ml with a concentration range of 2x10-4-1x10-6 M in 0.1% DMSO. Mixing was achieved by swirling the dish and the number of thrashes produced each minute was counted at regular intervals.

Figure 33: Analysis of locomotion in liquid.

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Michelle Joyner Results 1 3.4.2. Reversibility of compound effects in liquid.

The reversibility of compound effects was investigated by removing worms from the compound under test (typically after 60 min exposure by which time the inhibition had reached a maximal and stable level) into an excess of M9 buffer without compound. Thrashing was then measured at intervals up to 40 min after removal.

3.5. Analysis of locomotion on solid (agar) medium; body bends

To measure locomotion on solid medium (agar), the number of forward body bends generated by individual worms over a minute was counted. A forward body bend was defined as the movement in which the head of the worm moves to create a bend and then repeats to generate a bend that travels from head to tip of tail so completing a full sinusoid. Each body bend was counted from the head of the worm.

3.5.1. Dosing for body bends assays; in food (figure 34)

The food spot on the NGM agar plates was dosed with compound as follows: 5 cm plates were seeded with OP50 and left for 48h. Next 200 µl compound or vehicle was pipetted onto the OP50 lawn. Plates were dried for 1 hour and L4 + 1 day worms were transferred onto them and incubated at 20 °C for up to 2 hours. Individual worms were then removed to NGM plates of the same composition but with no bacterial lawn for one minute to remove any OP50 that had adhered to the worm and prevent transfer of OP50 to the assay plate. Cleaned worms were then individually transferred to a final unseeded drug free plate and allowed to equilibrate for 1 minute before counting the number of forward body bends produced by each worm in one minute.

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Michelle Joyner Results 1 To determine the optimal exposure time for the assessment of the effects of each compound on the locomotion of C. elegans on food- dosed agar plates the time-course of the effect of the maximum test concentration, 1 x10-4 M, was followed up to 2 hours after initial exposure.

Worms were assayed for effects on locomotion, following exposure for 15, 30, 45, 60, 90 and 120 minutes.

In subsequent experiments young adult (L4+1) worms were picked onto compound/control plates and incubated at 20 °C for two hours.

Figure 34: Analysis of locomotion on ‘food-dosed’ plates.

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Michelle Joyner Results 1 Inter Assay Variability for food-dosed body bends assays

Locomotion assays undertaken on different days were compared for inter-assay variability. ‘Food-dosed’ plates with a final concentration of 100 µM compound or 1% vehicle were used. Worms were picked onto either compound or vehicle control plates and incubated for two hours at 20°C before scoring the number of body bends produced per minute in individual worms.

Tribendimidine stability

Tribendimidine is reported to be rapidly metabolised in the host (Guo et al. 2009) and therefore the possibility that it may also be degraded by the OP50 bacterial lawn was investigated. Plates were ‘food-dosed’ with a stock solution of tribendimidine to give a predicted final concentration of 100 µM based on the assumption that the compound had diffused equally through the volume of agar in the plate. Plates were dosed ½, 2, 6 and 24 hours prior to use. Adult worms (L4 + 1 day) were incubated at 20 °C on compound/vehicle plates for 2 hours before counting the number of body bends produced per minute.

3.6. Dosing for body bends assays; in agar (figure 35).

Agar NGM was prepared and allowed to cool to 60 °C before adding compound to liquid NGM, with stirring, in a 1:10 dilution, the final vehicle concentration, when used, was 0.1% unless otherwise stated. This method is referred to as ‘agar-dosed’. Assay plates were used within one week of pouring and were stored at room temperature, wrapped in foil. Prior to use they were seeded with OP50 according to the standard protocol.

L4 + 1 day worms were transferred to these plates and incubated at 20 °C for 2 hours. Individual worms were then transferred to an agar-dosed 117

Michelle Joyner Results 1 plate with no food for 1 minute and then to a final un-seeded, agar- dosed plate, for 1 minute before counting the number of body bends produced in 1 minute.

Figure 35: Analysis of locomotion on ‘agar-dosed’ plates.

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Michelle Joyner Results 1 3.7. Results

An overview of different dosing regimes

A significant, dose-dependent inhibition of locomotion was observed with each of the methods used (liquid, food or agar dosing) for all of the compounds (figure 36).

A comparison of the relative potency across the three dosing methods indicates that liquid dosing has the most marked effect on inhibition of motility for all the compounds, followed by agar-dosing with food- dosing being least effective.

Based on these findings the concentration-dependence and kinetics of drug action was investigated more precisely by using the liquid dosing and the thrashing assay.

Figure 36: A Comparison of the locomotory inhibition by cholinergic compounds of each compound with ‘food-dosed’ , ‘agar- dosed’ plates or liquid dosing (next page). The inhibition of locomotion in worms on (A, E & I) tribendimidine, (B, F & J) amidantel, (C, G & K) Bay d9216 & (D, H & L) levamisole. Top row - inhibition of body bends produced after 2 hours on food-dosed plates. Middle row - the inhibitory effects of cholinergic compounds produced on agar-dosed plates. Bottom row – inhibitory effect on the thrashing rate of worms in buffer with compound of interest. Data points are the mean (±se) of n≥18 worms (≥3 experiments, and 6 worms per experiment). One-way ANOVA with Bonferroni post-hoc test *** p <0.001, ** p <0.01, * p <0.05 compared to control.

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Tribendimidine Amidantel Bay d9216 Levamisole (A) (B) (C) (D)

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Michelle Joyner Results 1 3.8. Dose-dependence, time-course and reversibility of inhibitory effects with liquid dosing.

Levamisole:

The thrashing rate of C. elegans exposed to 100 or 200 µM levamisole was completely inhibited in thrashing within 1 minute of addition of the compound and those exposed to 10 µM were completely inhibited within 10 minutes (figure 37) Inhibition of thrashing was sustained throughout the exposure period, up to 60 minutes. Worms exposed to 1 µM levamisole were significantly inhibited in their thrashing behaviour for the first 7 minutes of exposure when the thrashing rate of these worms was reduced by an average 50% of control worms. Effects were variable and worms recovered to the control thrashing rate during the exposure period. On removal to an excess of drug free buffer no reversal of the effects of 10 µM levamisole or above was seen. Worms exposed to 1 µM levamisole thrashed at or near the same rate as control worms when removed to drug free buffer (figure 37).

Tribendimidine

Tribendimidine showed a similar rapid time course and dose dependent inhibition as levamisole. Addition of 10 – 200 µM tribendimidine demonstrated a complete inhibition of locomotion within three minutes of application (figure 38). Worms exposed to 1 µM tribendimidine showed a 50% inhibition but variation prevented this trend from reaching significance. The effect of tribendimidine was slowly and partially reversible for 10 µM but irreversible for 100 µM (figure 38).

Bay d9216

Bay d9216 effected a rapid and dose dependent inhibition of thrashing (figure 39). Exposure to concentrations greater than 10 µM Bay d9216 121

Michelle Joyner Results 1 showed > 90% inhibition within 3 minutes. Complete inhibition of thrashing was seen within 10 minutes of addition of Bay d9216 and remained so for the 1 hour they were exposed to compound. 10 µM Bay d9216 reduced the thrashing rate of exposed worms to <10% of control within 3 minutes, their thrashing rate then fluctuated between <10% and ~15% of control worms for 30 minutes after addition of compound. The thrashing rate at this concentration was inhibited by 75% and remained in a steady state after 30 minutes exposure. The average thrashing rate of worms exposed to concentrations of lower than 10 µM showed clear inhibition but the variability in these lower doses precluded the observations reaching statistical significance.

Reversal of Bay d9216 effects was assessed by removing worms to an excess of buffer and scoring the thrashing rate at regular intervals for a further 40 minutes. Worms exposed to 1 µM Bay d9216 did not recover and in fact became significantly more inhibited in their thrashing behaviour 25 minutes after removal to drug-free buffer.

The thrashing rate of worms previously exposed to 100 µM increased from complete inhibition to 70% of control within 10 minutes after removal to buffer. This level of recovery was not sustained and the thrashing rate of these worms although still higher than in the presence of compound showed inhibition 30 minutes after removal to buffer. Reversal of the effects of 200 µM Bay d9216 was slower than those exposed to 100 µM, but this recovery of thrashing was more sustained, 10 minutes after removal to drug-free buffer worms were thrashing at 15% of the rate of control worms, recovery of thrashing continued to 60% of the rate of control worms, this dropped at the final time point to 50% compared to control (figure 39).

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Drug on Drug off

Figure 37: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM levamisole.

Data points are the mean (±se) of n≥6 experiments. Significance tested relative to 0.1% DMSO using the two-way ANOVA with Bonferroni post-hoc test, * p <0.05, ** p <0.01, *** p <0.001.

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Drug on Drug off

Figure 38: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM tribendimidine. Data points are the mean (±se) of n≥6 experiments. Significance tested relative to 0.1% DMSO using the two-way ANOVA with Bonferroni post-hoc test, * p <0.05, ** p <0.01, *** p <0.001. All points below horizontal line = ***.

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Drug on Drug off

Figure 39: The inhibition and recovery of thrashing in worms exposed to 1- 200 µM Bay d9216. Data points are the mean (±se) of n≥6 experiments. Significance tested relative to 0.1% DMSO using the two-way ANOVA with Bonferroni post-hoc test, * p <0.05, ** p <0.01, *** p <0.001. All points below horizontal line = ***.

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3.9. Estimating the relative potency of compounds

Maximum inhibition of thrashing was reached within 3 minutes of exposure to >10-6 M tribendimidine and Bay d9216, and within 10 minutes for worms exposed >10-6 M levamisole. Steady state of inhibition was estimated by taking the thrashing rate 30 minutes after drug exposure. A dose response curve for each compound was generated from the steady state inhibition and plotted relative to the thrashing rate after 30 minutes in vehicle control (figure 40). From these data the IC for tribendimidine was 1 µM (fitted to the modified logistic 50 equation, GraphPad Prism; 95% confidence intervals 0.6 µM – 2 µM; n=6), 4 µM for Bay d9216 (fitted to the modified logistic equation, GraphPad Prism; 95% confidence intervals 2.5 µM – 7 µM; n=6) and 3 µM for levamisole (fitted to the modified logistic equation, GraphPad Prism; 95% confidence interval 2 µM – 3.5 µM; n=6).

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100 Tribendimidine Bay d9216 Levamisole

50 % inhibition %

0 -8 -7 -6 -5 -4 -3 Log [compound] M

Figure 40: Dose response curve showing the percentage inhibition of thrashing in worms exposed to 1 – 200 µM tribendimidine, Bay d9216 or levamisole in liquid at steady state inhibition. Dose response curves based on the percentage of thrashes per minute relative to control (0.1% DMSO) worms after 30 minutes exposure to each compound in buffer when a steady state of inhibition was reached.

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3.10. An analysis of the kinetics of compound effects

The time taken to reach 50% of the maximal inhibition (t ½ on) for each compound in the concentration range 10 – 200 µM was determined. From these data it is clear that in worms exposed to concentrations greater than 1 µM of each compound the onset of inhibition was very rapid. Worms exposed to 10 µM tribendimidine took an average of 90 seconds to reach t ½ on whereas worms exposed to the same concentration of levamisole took an average of 40 seconds to reach t ½ on. Bay d9216 was the fastest acting compound at this concentration and took 30 seconds to reach t ½ on. The reversal of inhibitory effects was analysed (t ½ off rate) and no reversal of the effects of concentrations of 100 µM or above of tribendimidine or levamisole were observed. The t ½ off rate for 100 µM Bay d9216 was 7 minutes and 15 minutes for 200 µM Bay d9216. IC s and t ½ on and off rates are 50 summarised in table 9.

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t on t off Concentration 1/2 1/2 Compound IC M 50 M (Min) (Min)

1x10-4 0.5 NR Tribendimidine 1x10-6 2x10-4 0.5 NR

1x10-4 0.5 7 Bay d9216 4x10-6 2x10-4 0.5 15

1x10-4 0.5 NR Levamisole 3x10-6 2x10-4 0.5 NR

Table 9: Summarising the t on and off rate at maximum inhibition and 1/2 the IC at steady state inhibition for tribendimidine, Bay d9216 and 50 levamisole. NR = No significant reversal of compound effect

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3.11. Time-course for food dosing

The time-course for the effect of the compounds on body bends with food dosed plates was followed for up to two hours (figure 41) and indicates that the level of inhibition changes between 15 and 120 min for some of the compounds whilst for levamisole the level of inhibition is constant.

The observed changes in inhibition at different time points may be due to the compound diffusing through the agar so becoming less concentrated at the food spot. Alternatively this may be due to worms leaving the drug-laced food spot so altering the exposure time of individual worms. The possible reasons for these differences suggested here would be eliminated on agar-dosed plates where the compound would be uniformly distributed through the agar. This clearly demonstrates that agar-dosing is the optimum method when compared to food-dosing protocols.

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1% EtOH 1% DMSO

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Figure 41: Determination of optimal exposure time on food-dosed plates (previous page). Compound or vehicle was added to prepared NGM plates to give a final concentration of 100 μM or 1% vehicle. The mean (±SD) locomotion of adult wild type C. elegans on NGM agar plates measured at different time points is shown. Locomotion was analysed as body bends per minute. n≥12, 6 worms per experiment worms. (A) 1% EtOH, (B) 1% DMSO, (C) tribendimidine, (D) amidantel, both in 1% DMSO, (E) Bay d9216, & (F) levamisole both in 1% EtOH.

3.12. Inter Assay variability for food-dosed plates

The level of inhibition of body bends on food-dosed plates was similar between different assays conducted on different days for amidantel. For tribendimidine, Bay d9216 and levamisole the results were more variable. No significant difference was found between assays conducted on different days with any of the compounds when tested using the one- way ANOVA (figure 42).

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Figure 42: No significant inter-assay variability was seen between assays on different days. Mean (±SD) forward locomotion of C. elegans on ‘food-dosed’ agar plates was measured as body bends per minute following exposure to 100 µM (A) tribendimidine, (B) amidantel, (C) Bay d9216 or (D) levamisole. Each bar represents individual assays numbered 1-5. No significant difference was found when tested using the one-way ANOVA. n≥6 worms.

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3.13. The effect of the time after food-dosing on the inhibitory action of tribendimidine

The greatest effect was observed on plates on which the food had been dosed with compound just 30 min prior to addition of the worms (figure 43) whilst later addition of worms also resulted in an inhibition of locomotion but this was less marked and the same for plates 2 through to 24 hours after food dosing.

15 -1 10

5 Body Bends min Bends Body

***

0 DMSO 1/2 2 6 24 T hrs

Figure 43: Tribendimidine stability Mean (±SD) locomotion of C. elegans on ‘food-dosed’ plates after exposure to 100 µM tribendimidine or 1% vehicle for 2 hours. Worms were transferred to compound/vehicle plates ½, 2, 6 and 24 hours after addition of tribendimidine to the plate. Significance was determined relative to vehicle control using the one-way ANOVA. *** p <0.001; ** p < 0.01, n≥ 6 worms. All points below horizontal line = **.

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3.14. Discussion.

Dosing methods

Clear differences between the different dosage regimens support the conclusion that liquid dosing is the preferred route for testing the effects of compounds on C. elegans. This method provides robust and reproducible effects on locomotion in a dose-dependent manner. Furthermore, the highest level of inhibition is observed with this method of applying the compound to the worm. Worms on ‘food-dosed’ plates appeared to show the least potent inhibition indicated by the relative inhibition. This likely reflects that the effective concentration of compounds diluted into the food spot is an ineffective route to dosing worms. In particular this method relies on the worms remaining on the food spot. Under normal circumstances dwelling on food is the preferred state with food leaving occurring less than once in two hours. However addition of chemical compounds may increase the tendency of worms to leave food patches and so increase the time spent off food and this impacts on the level of inhibition observed.

The observation that dosing in liquid, a situation in which the worms do not feed and therefore are unlikely to ingest significant quantities of the compound, is more effective than dosing on food, a situation in which the worms will orally ingest the compound, is consistent with the suggestion that the compounds can readily cross the cuticle of the worm. This raises the question of the relationship between the external and internal drug concentration in the worm. Earlier investigations on the potency of levamisole in whole compared to cut worms in which the whole worm is approximately three orders of magnitude less sensitive indicates that the concentration is likely to be less internally (Tomlinson et al. 1985). There are no dose responses for levamisole on nAChRs on C. elegans body wall muscle from electrophysiological experiments with which to make a comparison. The EC obtained here for levamisole, 50

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3µM, is in reasonably close agreement with an earlier reported value for whole worms which was 9 µM. (Qian et al. 2008). The difference between these values is likely to be due to the fact that the published EC refers to paralysis (complete lack of movement or coiling) whereas 50 the value derived here refers to a lack of thrashing movements but not necessarily total paralysis.

No difference in inhibition of locomotion was observed between vehicle controls on ‘food-dosed’ (1% DMSO) and ‘agar-dosed’ (0.1% DMSO) plates (figure 36). In a comparison of DMSO effects in thrashing there was no difference observed between the thrashing rate of worms in buffer and that of worms in buffer supplemented with 1 or 0.1% DMSO (data not shown). Although dosing the agar provides a better route to defining drug concentration particularly if drug accumulation is readily achieved through the cuticle there appeared to be some limit to drug potency using this methodology. This is particularly clear where levamisole concentrations which give a full paralysis in liquid only partially inhibit worms on agar. This implies that dose dependence defined by this approach is of limited value. In contrast the approach in which thrashing in drug free and drug laced medium are compared showed a more robust effect. For all compounds exposing the organism to compound in liquid caused a dramatically higher level of inhibition of locomotion compared to NGM assays.

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Locomotion.

Tribendimidine, amidantel, Bay d9216 and levamisole showed inhibition of locomotion on both agar and in liquid. This is in agreement with the previously reported mode of action of these compounds as agonists of nAChRs at the worm NMJ (Lewis et al. 1980; Tomlinson et al. 1985).

In experiments using ‘food-dosed’ agar plates the maximal inhibition of body bend generation was approximately 50% with doses of 100 µM. These experiments were confounded by the dosing method used where the compound was added to the food spot in the plate so the final concentration was an estimate. When the worm left the food spot it was no longer exposed to the compound meaning that all the worms tested did not experience the same dose of compound. The use of ‘agar-dosed’ plates eliminated these confounds by increasing the reproducibility of stated doses and providing a more uniform environment where worms on the same plate were more likely to be exposed to the same amount of compound. On ‘agar-dosed’ plates the maximal inhibition of body bends on 100 µM tribendimidine was 80% and 60% on 100 µM amidantel, Bay d9216 and levamisole.

The use of thrashing assays has also enabled the in depth investigation of the onset and reversal of drug effects. This type of close analysis may reveal subtle differences between the mechanisms of action of these compounds. The onset of effect was very rapid for tribendimidine, Bay d9216 and levamisole. These results show that the acetanilide compounds are comparable to levamisole in their onset of action and the level inhibition of locomotion in C. elegans.

The reversal of drug effects was investigated and it has been shown that the effects of levamisole were not reversed when worms were removed to an excess of buffer, this was also the case for worms exposed to concentrations of 100 µM tribendimidine or above. The effects of Bay d9216 were partially reversed and a recovery of approximately 50% of 137

Michelle Joyner Results 1 thrashing rate relative to control was observed in worms exposed to 100 µM Bay d9216 or above. The reversal of the effects of lower doses of Bay d9216 were complex, with an increase in inhibition after worms exposed to 1 µM were removed to drug free buffer.

3.15. Summary

In conclusion these investigations verify the value of C. elegans as a tool to investigate the anthelmintic potential of test compounds. The similar potencies of related compounds in the inhibition of locomotion support the theory that the acetanilide compounds and levamisole act as cholinergic compounds at the NMJ.

The methodology described in this chapter provides a tractable route to investigate candidate cholinergic receptors in the action of the acetanilide compounds. The evaluation of the different dosing protocols has shown that the most efficacious protocol in which to test the inhibitory effects of these compounds is with dosing in liquid. This has allowed the investigation of concentration dependence with each compound and the analysis of the time course of compound effects.

Despite previously reported differences in the ability of amidantel and Bay d9216 to exert their effects in whole and cut worms (Tomlinson et al. 1985) the findings presented here show that the acetanilides effectively cross the cuticle and inhibit locomotion in the whole worm in a manner to comparable levamisole.

Tribendimidine has been reported to act through the same subtype of nAChRs as levamisole (Hu et al. 2009). The recovery analysis in this chapter has revealed interesting differences in the recovery of worms which had been previously exposed to tribendimidine and levamisole where no recovery was seen compared those exposed to Bay d9216 which recovered to approximately 50% of control. This suggests that Bay

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Michelle Joyner Results 1 d9216 may be acting through a different subtype of nAChR to levamisole.

Further investigations into the effects of the acetanilides on behaviours other than locomotion in wild type worms will be compared to the effects of levamisole in the following chapter. Further differences in the behavioural effects of these compounds will elucidate whether or not any of the acetanilides have a levamisole resistance breaking potential.

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Michelle Joyner Results 2 Chapter 4. A comparison of the effects of levamisole, amidantel, Bay d9216 and tribendimidine on C. elegans behaviours and development

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Michelle Joyner Results 2 4.1. Introduction

In order to assess whether or not there are any differences in the mode of action of the compounds a comparison has been made between the effects of all the compounds on a range of C. elegans behaviours. The effects of the compounds on motility are reported in chapter 3 as these assays were used to establish optimal dosing. This detailed pharmacological analysis of the effects on thrashing permitted the design of drug dosing protocols and a broader investigation of the effects the compounds on a range of behaviours encompassing feeding (pharyngeal pumping) , egg-laying (regulation of vulval muscle contraction), reproduction (progeny number) and development. Assays for progeny number encompass the processes of egg production and fertilization whilst assays for development encompass hatching and larval moults.

There are no previous reports on the effects of the acetanilide compounds on pharyngeal pumping. Mutations in the gene encoding the levamisole sensitive subunit LEV-8 exhibit a reduced pumping rate (Towers et al. 2005) and high concentrations of levamisole inhibit pharyngeal pumping in wild type C. elegans (Lockery et al. 2012)

In the absence of drug, unmated wild type C. elegans produce ~ 300 progeny (Singson 2001). Tribendimidine and levamisole have been reported to cause a reduction in the number of progeny produced by worms exposed to these compounds from the L4 larval stage (Hu et al. 2009). Amidantel and Bay d9216 are reported to delay the onset of egg laying and reduce the number of eggs laid by wild type C. elegans exposed either compound from the first larval stage (Woods et al. 1986).

Tribendimidine and levamisole are both reported to stimulate egg laying (Trent et al. 1983; Kim et al. 2001; Hu et al. 2009). There are no reports on the acute effect of amidantel or Bay d9216 on egg laying behaviour

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Michelle Joyner Results 2 in C. elegans. Amidantel and Bay d9216 are reported to have no effect on the timing of development in C. elegans (Woods et al. 1986). Tribendimidine is reported to inhibit larval development (Hu et al. 2009). The levamisole sensitive subunit UNC-63 has been implicated in the neuronal control of developmental timing (Ruaud and Bessereau 2006).

4.2. Methods

4.2.1. Pharyngeal pumping

C. elegans feed by rhythmically contracting the pharyngeal muscle so pumping bacteria through the pharynx to the intestine. On food the average wild type pharyngeal pumping rate is ~ 250 times per minute (Towers et al. 2005). This can be visualised under a dissection microscope. A pump was defined as the movement of the pharynx up and back to its original position. To study the effect of the compounds on pharyngeal pumping it was necessary to carry out the assays using the food-dosed paradigm for drug exposure as pharyngeal pumping behaviour is markedly reduced in the absence of food (Avery and You 2012).

Therefore, to determine the effect of the compounds on the rate of pharyngeal pumping, young adults (L4 + one day old) were exposed to 10-100 µM of each compound on food-dosed plates for 2 hours at 20 °C before counting the number of pumps produced per minute by each worm while on food. The pumping rate was compared to that of 1% vehicle control worms conducted in parallel. The lowest dose tested, 10 µM, and the time of exposure, 2 hours, had previously been established as causing effective inhibition of locomotion (chapter 3).

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Michelle Joyner Results 2 4.2.2. Reproduction and survival; Number of progeny per worm

To measure the effects of compounds on the number of offspring produced per worm a microtitre plate protocol based on a published method (Hu et al. 2009) was adopted. For this a single L4 C. elegans was pipetted into each well of a 48 well micro-titre plate which was prepared with 160 μl M9/0.1% BSA, 20 μl OP50 (OD =3) and 20 μl compound in 600 solution to give a final volume of 200 μl with a concentration range of 2x10-4 - 5x10-3 M in 0.1% DMSO (figure 44). The plates were sealed, wrapped with foil to exclude light and incubated at 20 °C for three and a half days (84 hrs.). Three replicate wells were prepared for each concentration. The contents of each well was then pipetted onto a non- seeded (no drug) plate, one plate per well and the total number of offspring were immediately counted. Survival of offspring was determined based on response to touch and colouration. All results were compared to 0.1% DMSO. This time period represents approximately half the egg laying cycle in wild type worms so in the absence of drug approximately 150 progeny would be expected, reflecting the fact that the entire egg laying stage was not investigated.

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Figure 44: Analysis of brood size

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Michelle Joyner Results 2 4.2.3. Egg laying behaviour C. elegans Adult hermaphrodite worms (up to 3 days old) lay eggs in the presence of food. The rate of egg-laying is regulated by neural circuitry driving the activity of the vulval muscle and includes excitatory cholinergic neurons (Kim et al. 2001). The stimulatory effects of tribendimidine on egg laying in C. elegans have previously been reported (Hu et al. 2009). In order to determine the effect of Bay d9216 on egg laying seeded ‘agar-dosed’ plates were used as worms only lay eggs in the presence of food. The effects of Bay d9216 on egg laying behaviour were compared to those of levamisole (figure 45). A single gravid adult (L4+2) was placed onto seeded agar plates containing 10 – 100 µM Bay d9216 or levamisole. The adult was removed after incubation for 1 hour at 20°C and the number of eggs laid under each condition was scored and compared to NGM control.

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Figure 45: Analysis of egg laying effects

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Michelle Joyner Results 2 4.2.4. Developmental assays; Timing of development ‘Agar-dosed’ plates seeded with OP50 were used for developmental assays. The final concentration range was 10-3 M, 2 x 10-4 M or 10-4 M in 0.1% DMSO or no vehicle as stated. Freshly laid eggs were picked from non-drug plates and transferred to compound or control plates (0.1% DMSO or NGM only). Plates were prepared in triplicate for each condition. Plates were sealed with parafilm and wrapped in foil before incubating at 20 °C. Hatched and unhatched eggs and the number of worms at each developmental stage were scored at 24, 48 & 72 hours. Worms were not transferred to fresh plates during the assay period as the food source was not exhausted on any of the plates.

4.3. Results 4.3.1. Pharyngeal pumping

Worms incubated on food-dosed plates with 10 µM tribendimidine, amidantel, Bay d9216 or levamisole were not affected in their pumping behaviour relative to control (figure 46) which suggests relative refractory of the pharyngeal NMJ to this action. Thus the effects of the compounds at a higher concentration of 100 µM were also investigated (figure 47). In these experiments worms exposed to 100 µM levamisole were inhibited in their pharyngeal pumping rate. The same dose of Bay d9216 had no effect on pumping behaviour.

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(A) 250 NSD (B) 250 NSD

200 200

-1 -1 150 150

100 100

Pumpsmin Pumpsmin 50 50

0 0

-5 M tri -5 M ami 10 1% EtOH 1% DMSO 10 (C) (D) 250 NSD 250 NSD

200 200

-1 -1 150 150

100 100

Pumpsmin Pumpsmin 50 50

0 0

-5 M lev -5 M Bay 1% EtOH 1% EtOH 10 10

Figure 46: Comparison of the effects of tribendimidine, amidantel, Bay d9216 or levamisole on pharyngeal pumping rate. Worms were incubated on 10 µM ‘food-dosed’ plates for 2 hours at 20°C before counting the pharyngeal pumping (pumps per minute). (A) Tribendimidine, (B) amidantel, (C) Bay d9216 or (D) levamisole. No significant effect was seen when tested using the one-way ANOVA with Bonferroni post-hoc test. Horizontal lines are the mean average (±se) of n≥10.

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NSD 250 ***

200 -1 150

100 Pumpsmin 50

NGM -5 M lev -4 M lev -5 M Bay -4 M Bay 10 10 10 10

Figure 47: The effects of 10 – 100 µM Bay d9216 or levamisole on pharyngeal pumping rate. Agar-dosed plates dosed with 10 - 100 µM Bay d9216 or levamisole. 100 µM levamisole significantly inhibited pumping. Bay d9216 had no effect on pumping behaviour. Significance tested with one-way ANOVA with Bonferroni post-hoc test, *** p <0.001 compared to control. Horizontal lines are the mean average (±se) of n≥10.

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Michelle Joyner Results 2 4.3.2. Reproduction

Tribendimidine, Bay d9216 and levamisole reduced the number of progeny produced by worms exposed to the highest concentration. In the case of Bay d9216 brood size was only reduced at the highest concentration tested and had no effect at lower doses. In contrast both levamisole and tribendimidine showed a dose dependent reduction in brood size from concentrations above 50 µM and at the highest concentration used brood size was reduced by 90% for both compounds (figure 48).

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(A) 200 150 100 50 *

No. progeny No. ** 0

-5 -4 -4

5 x 10 1 x 10 2 x 10 0.1% DMSO [Tri] M

(B) 200 150 100 **

50 No. progeny No. 0

-5 -4 -4

5 x 10 1 x 10 2 x 10 0.1% DMSO [Bay] M (C) 200 150

100 * 50

** No. progeny No. 0

-5 -4 -4

5 x 10 1 x 10 2 x 10 0.1% DMSO [Lev] M

Figure 48: The effects of Bay d9216, tribendimidine or levamisole on the number of progeny produced by C. elegans adults

Individual L4 worms were placed into the wells of a 48-well microtitre plate containing 50 – 200 µM of compound in M9/BSA buffer and E. coli OP50 (OD =3) as shown. After 3.5 days at 20°C the contents of each 600 well was tipped into an agar plate and the numbers of offspring produced by worms exposed to (A) tribendimidine, (B) Bay d9216 or (C) levamisole were scored. Data points are the mean brood size (±se) of ≥3 replicates. Significance tested with one-way ANOVA with Bonferroni post-hoc test, * p < 0.05, ** p < 0.01.

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Michelle Joyner Results 2 4.3.3. Egg laying

At the highest dose both Bay d9216 and levamisole had a stimulatory effect on egg laying behaviour relative to control worms (figure 49). 100 µM levamisole had the greatest stimulatory effect, increasing the number of eggs laid in an hour by over 100% relative to control. The same concentration of Bay d9216 had less of an effect and increased the number of eggs by just over 50% of the number of eggs laid by control worms. No effect was seen in worms on 10 µM plates compared to those on NGM.

*** -1

15 **

5 Number eggs laid hour laid eggs Number

NGM

-5 M Bay -4d9216 M Bay -5d9216 M levamisole-4 M levamisole 10 10 10 10

Figure 49: Effects of exposure to Bay d9216 or levamisole on the egg laying behaviour of C. elegans ‘Agar-dosed’ plates were used to test the effects of 10 - 100 µM Bay d9216 or levamisole on the egg laying rate of adult worms. Data points are the mean (±se) of n≥10. Significance tested with one-way ANOVA with Bonferroni post-hoc test, **p <0.01, *** p <0.001.

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Michelle Joyner Results 2 4.3.4. Development

No differences in developmental timing were observed between the two control conditions (0.2% DMSO or NGM). A small number of eggs failed to hatch on 200 µM tribendimidine plates and this number was slightly increased on the 1 mM plates. Of the eggs that hatched, tribendimidine had no effect on developmental timing over 3 days (figure 50).

Similar results were obtained with amidantel where a smaller number of eggs failed to hatch on 1 mM plates (figure 51). A small number of worms failed to reach adult stage within 72 hours on the highest concentration, these went on to develop to adult worms within a further 24 hours (data not shown).

Comparable results were obtained with Bay d9216 (figure 52). No effect on the timing of development was observed with any of the concentrations tested and a small number of eggs failed to hatch on 200 µM plates but not on 1 mM plates. A very small number of worms on 1 mM plates showed delayed development from L4 to adult stage, all had reached adulthood within 96 hours (4 days).

Levamisole had no effect on timing of development or egg hatching at 100 µM. A small number of eggs failed to hatch on 200 µM plates, of the hatched eggs all reached adult stage within 72 hours. 1 mM levamisole plates had a slightly larger number of un-hatched eggs compared to lower concentrations or the other compounds and a delay in developmental timing was observed, at 48 hours exposure all worms were at the L4 stage on NGM control plates, on 1 mM levamisole there was a mixture of L1, and L2 or L3 larvae alongside the L4 larvae which comprised the majority of the population. At 72 hours exposure the majority of worms were at the L4 stage, control worms were all adults within 72 hours. ~ 25% were adults and very few were still at the L3

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Michelle Joyner Results 2 larval stage (figure 53). All worms reached adult stage within 4 days (data not shown).

The effects of each compound on the timing of development are summarised in figure 54 which shows the percentage of the population which reached the adult stage after 72 hours exposure from egg. This shows that amidantel had no effect on the timing of development, tribendimidine had a small, but significant effect at the highest concentration tested. Bay d9216 had no effect on the timing of development at the lowest and highest concentrations tested, at 200 µM Bay d9216 the number off eggs reaching adult stage was decreased compared to control, however these plates contained an increased number of un-hatched eggs rather than developmentally delayed worms. Levamisole delayed the timing of development in a dose dependent manner. No effect was seen in larvae developing on 100 µM levamisole, an increasing effect was seen on 0.2 – 1 mM levamisole.

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Day 1 Tribendimidine 0.2% DMSO 100 M 200 M 1 mM 50 50 50 50

25 25 25 25

No. worms

No. worms No. worms 0 worms No. 0 0 0

L1 L4 Egg L2/3 L1 L4 L1 L4 L1 L4 Adult Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms No. worms No. worms No. No. worms No. 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms No. worms No. worms No. No. worms No. 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Day 3 Developmental stage Developmental stage Developmental stage Developmental stage

Figure 50: A comparison of the effect of tribendimidine on the timing of development.

Tribendimidine was added to NGM to give final concentrations of 100 µM, 200 µM & 1 mM in 0.2% DMSO. Eggs were placed onto the plates and allowed to develop over 3 days. The effect of tribendimidine on the timing of development was compared to DMSO control. Data points are the mean (±se) of n≥50 worms (≥4 experiments).

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Day 1 Amidantel 0.2% DMSO 100 M 200 M 1 mM 50 50 50 50

25 25 25 25

No. worms

No. worms No. worms No. worms 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms

No. worms No. worms No. worms 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms No. worms No. worms No. worms 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Day 3 Developmental stage Developmental stage Developmental stage Developmental stage

Figure 51: A comparison of the effect of amidantel on the timing of development.

Agar-dosed plates were used to assess the effect of 100 µM, 200 µM & 1 mM amidantel on developmental timing. Eggs were placed onto the plates and allowed to develop over 3 days. Data points are the mean (±se) of n≥50 worms (≥ 4 experiments).

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Day 1 Bay d9216 NGM 100 M 200 M 1 mM 50 50 50 50

25 25 25 25

No. worms No. worms No. worms 0 0 0 worms No. 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult

Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms No. worms No. worms No. worms 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult

Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. worms No. worms No. worms No. worms 0 0 0 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Day 3 Developmental stage Developmental stage Developmental stage Developmental stage

Figure 52: A comparison of the effect of Bay d9216 on the timing of development.

Agar-dosed plates were used to assess the effect of 100 µM, 200 µM & 1 mM Bay d9216 on the timing of development. Eggs were placed onto the plates and allowed to develop over 3 days. Data points are the mean (±se) of n≥50 worms (≥ 4 experiments).

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Day 1 Levamisole NGM 100 M 200 M 1 mM

50 50 50 50

25 25 25 25

No. No. worms No. worms No. worms 0 0 0 No. worms 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. No. worms No. worms No. worms 0 0 0 No. worms 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult

Developmental stage Developmental stage Developmental stage Developmental stage

50 50 50 50

25 25 25 25

No. No. worms No. worms No. worms 0 0 0 No. worms 0

L1 L4 L1 L4 L1 L4 L1 L4 Egg L2/3 Egg L2/3 Egg L2/3 Egg L2/3 Adult Adult Adult Adult Day 3 Developmental stage Developmental stage Developmental stage Developmental stage

Figure 53: A comparison of the effect of levamisole on the timing of development.

Agar-dosed plates were used to assess the effect of 100 µM, 200 µM & 1 mM levamisole on the timing of development. Data points are the mean (±se) of n≥50 (≥4 experiments).

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(A) (B) NSD 100 100 * 80 80

60 60

40 40

20 20

%Population atstage Adult %Population at Adult stage %Population at Adult

0 0 -4 -4 -3 0.2% DMSO 10-4 2 x 10-4 10-3 0.2% DMSO 10 2 x 10 10 Log [tribendimidine] M Log [amidantel] M

60 60

40 40 ***

20 20

%Population stage at Adult %Population stage at Adult

0 0 -4 -4 -3 NGM 10-4 2 x 10-4 10-3 NGM 10 2 x 10 10 Log [Bay d9126] M Log [levamisole] M

Figure 54: The percentage of worms which reached adult stage when allowed to develop on agar-dosed plates (seeded with OP50).

The percentage of the population that reached adult stage following exposure from egg for 72 hours on 0.1 – 1 mM (A) tribendimidine, (B) amidantel, (C) Bay d9216 or (D) levamisole. Data points are the mean (±se) of n≥50, ≥4 experiments. All points below horizontal line = NSD.

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4.4. Discussion.

The previous chapter (chapter 3) provided evidence that all of the compounds being investigated exert an inhibition of motility over a dose range very similar to that of levamisole. In this chapter this has been extended to compare the effects of the compounds with that of levamisole in further assays of neuromuscular function which are involved in feeding behaviour and egg-laying. In addition the effect on development has been investigated. The dosing regimens used were based on the results obtained in chapter 3. A further microtitre plate assay was also deployed as this had advantages for studying brood size from individual worms.

Pharyngeal pumping

Of the compounds tested, only levamisole inhibited pharyngeal pumping and then only partially and at a concentration at least 10 fold higher than required to inhibit motility. Therefore the nAChRs that regulate the activity of the pharyngeal circuit are pharmacologically distinct from those that regulate locomotor behaviour. Furthermore, the observation that only levamisole had an effect indicates this compound has a pharmacological profile which is distinct from the other compounds tested.

Development and progeny

Developmental and progeny assays were undertaken following reports on the actions of tribendimidine (Hu et al. 2009). Methods used for the progeny assay were replicated according to these published reports (Hu et al. 2009). The results from this type of assay provides a readout of an integration of processes such as oocyte and sperm production, fertilisation, embryogenesis, egg laying and hatching, as well as developmental timing and larval moults.

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To investigate these different aspects further assays were designed. The reduced number of progeny was not due to a reduction in egg laying behaviour as these compounds have been shown previously to stimulate egg laying (Kim et al. 2001; Hu et al. 2009). In this chapter a stimulation of egg laying was also shown for Bay d9216 and levamisole.

In a distinct approach the transition from egg to adult was investigated as the number of progeny produced per worm could be affected by arrested development. Thus development through the different larval stages was systematically scored from egg to adult. No significant effect on egg hatching was observed at the highest concentrations tested. Interestingly tribendimidine amidantel and Bay d9216 did not affect the timing of development at the concentrations used to investigate effects on brood size. Similarly levamisole caused a minimal delay in developmental timing at concentrations of 200 µM. Therefore the effect of reduced progeny is not likely to be a developmental effect from the egg to adult stage.

The effects of the compounds on developmental timing patterns were investigated. Amidantel had no effect on egg hatching or developmental timing. When exposed to tribendimidine from egg onwards an increasing number of eggs failed to hatch with increasing concentration, however this effect was not significant. Developmental timing was not significantly delayed except for the highest concentration tested (1 mM) and this was due to the number of eggs which failed to hatch, of the hatched larvae, all developed to adult stage within the same timeframe as control worms. A similar observation was made with Bay d9216 where a significant number of eggs failed to hatch on 200 µM Bay d9216, but all hatched larvae developed normally. No hatching effect was observed on higher concentrations of Bay d9216. These observations may reflect effects via a Bay d9216 sensitive receptor which is desensitised at higher doses. Desensitisation of nAChRs at high

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Michelle Joyner Results 2 doses has been reported in body wall muscle of the parasite Ascaris suum (Pennington and Martin 1990; Robertson and Martin 1993).

Levamisole was the only compound to cause a significant and dose dependent delay in the timing of development, all larvae developed to the adult stage albeit slower than control worms or those exposed to the other compounds, this may be due to a disruption of the regulation of developmental timing, the nicotinic agonist DMPP is reported to slow development in C. elegans and this effect is mediated through levamisole sensitive UNC-63 containing neuronal receptors (Ruaud and Bessereau 2006). However the effects of levamisole were not lethal as is reported with DMPP therefore the slowed development observed at high concentrations of levamisole may also be due to a reduction of feeding behaviour since levamisole reduces the pharyngeal pumping rate at concentrations over 10µM. Eggs were placed close to the food source in all cases so this is unlikely to be due to paralysed worms being unable to reach food.

Death, based on response to touch and colouration, was not observed at the concentrations tested so the reduction of progeny number is not an effect of survival.

Sperm production occurs at the L3 stage i.e. prior to the exposure of the worm to drug so an effect on sperm production is unlikely to provide an explanation for the reduction in brood size. Thus it would seem that neither effects on sperm production, egg hatching, developmental transition from egg to adult stage nor nematocidal activity provide an adequate explanation for the reduced brood size.

Egg production occurs later than sperm production, at L4 (Nayak et al. 2005) and therefore drug exposure could impact on this process. Furthermore, oocyte maturation, fertilisation and embryogenesis will also occur during the period of drug exposure and the drugs may impact on any of these to reduce progeny number (figure 55). 163

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Figure 55: Potential sites of action causing the reduction in progeny number in wild type worms exposed to tribendimidine, Bay d9216 or levamisole

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4.5. Summary

In the previous chapter the similar potencies of related compounds in the inhibition of locomotion support the theory that the acetanilide compounds and levamisole act as cholinergic compounds at the NMJ. In this chapter further assays have been deployed which reveal some differences between the actions of the compounds and levamisole and lend support to the hypothesis that they may exert their actions through different subtypes of nAChRs. In particular, the data suggest that the mode of action of levamisole is distinct to that of Bay d9216. In the previous chapter (chapter 3) the inhibition of locomotion in Bay d9216 exposed worms was shown to reverse on removal to drug free buffer and this reversal was not seen in worms previously exposed to levamisole or tribendimidine. Distinct effects on pharyngeal pumping were also observed where the pharyngeal NMJ was refractory to Bay d9216 effects whereas 100 µM levamisole inhibited pumping. Levamisole retarded developmental timing and this was not shown with Bay d9216 treated worms. In progeny assays only the highest concentration of Bay d9216 caused a reduction in the number of progeny in treated worms, whereas levamisole treated worms showed a reduction in progeny size at lower concentrations. In contrast the effects of tribendimidine on the reduction of brood size showed similar concentration dependence to those of levamisole.

The acetanilide compounds have shown interesting actions in the context of anthelmintic compounds (table 10). They have been shown to inhibit locomotion and impact on reproductive capability. They do not overtly effect feeding but with their major target being the NMJ and the competence to produce offspring they will profoundly affect the survival capability of worms. If this model impacts on parasites these compounds offer good potential as anthelmintics. The genetic tractability of C. elegans is utilised in the next chapter where a screen of

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Michelle Joyner Results 2 cholinergic mutants is described in order to extend these investigations and define the molecular target(s) of these compounds.

Body Thrashing Pharyngeal Egg Progeny bends pumping laying size

Levamisole Inhibited Inhibited Inhibited Stimulated Reduced IC = 3 µM 100 µM 100 µM 100 µM 50

Bay d9216 Inhibited Inhibited No effect Stimulated Reduced IC = 4 µM 50 100 µM 200 µM

Amidantel Inhibited Inhibited No effect ND ND IC = ND 50

Tribendimidine Inhibited Inhibited No effect ND Reduced IC = 1 µM 50 100 µM

Table 10: A summary of the effects of levamisole, Bay d9216, amidantel and tribendimidine on C. elegans. Locomotion was inhibited with all compounds tested at concentrations of 10 µM or greater. There was no nematocidal or egg hatching effect for any of the compounds at the concentrations tested. Pharyngeal pumping was only inhibited by 100 µM levamisole. No effect was seen with the acetanilides. Levamisole was the only compound to cause a significant and dose dependent effect on developmental timing. Levamisole and tribendimidine caused a significant and dose dependent reduction in brood size, 200 µM Bay d9216 caused a reduction in brood size and no effect was seen at lower doses.

ND = Not done

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Chapter 5. A Reverse genetic screen to identify C. elegans strains with altered susceptibility to amidantel, Bay d9216,

tribendimidine or levamisole.

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5.1. Introduction

In this chapter a molecular genetic strategy has been adopted in order to determine whether or not any of the compounds, and in particular Bay d9216, has a distinct mode of action from levamisole. Importantly, this will provide evidence to support whether or not the compounds have the potential to act as levamisole-resistance breaking anthelmintics. The study has focused on the inhibitory actions on the compounds on motility (body bends and thrashing) which is exerted through an interaction with the nAChRs present on the body wall muscle of the worm. As described in the introduction, these have been delineated into two distinct subtypes: The L-AChR has been shown to be sensitive to levamisole and ACh, but not nicotine and conversely the N- AChR has been shown to be sensitive to nicotine and ACh but not levamisole. These receptor subtypes have been demonstrated both in vivo and when heterologously expressed in X. laevis oocytes (Richmond and Jorgensen 1999; Boulin et al. 2008).

The question of the subunit composition of the L and N nAChRs was briefly considered in the introduction and will now be described in more detail.

A micro-array profiling strategy (MAPCel) has been used to detect the nicotinic receptor subunits expressed in C. elegans body wall muscle. Of seven subunits found to be enriched in muscle, five were the previously characterised L-AChR subunits (UNC-38, UNC-63, UNC-29, and LEV-1 & LEV-8). Two further subunits identified were ACR-16 and ACR-8. Ablation of acr-8 had no effect on the nicotinic current in electrophysiological analysis, whereas deletion of acr-16 led to the elimination of this current. Therefore The N-AChR is proposed to be a homomeric receptor which is comprised of five ACR-16 subunits (Touroutine et al. 2005).

This chapter describes the susceptibility of levamisole resistant C. elegans mutants to amidantel, tribendimidine and Bay d9216. These 168

Michelle Joyner Results 3 strains can be divided into three general groups defined by their phenotype. The ‘unc’ mutants move in an uncoordinated manner and are highly resistant to levamisole (Lewis et al. 1980). This group grows more slowly and produce fewer offspring than wild type worms (Lewis et al. 1980). Pseudo wild type worms move normally but are less sensitive to levamisole than wild type worm (Lewis et al. 1980). The third group are classed as 'twitchers', these mutants move normally except for twitches which occur spasmodically and are partially resistant to levamisole (Brenner 1973). The ‘uncs’ and pseudo wild type levamisole resistant mutants arise due to functionally deficient L-AChRs, whereas the 'twitchers' arise due to deficiencies downstream of the L-AChR (Lewis et al. 1980).

Levamisole resistant mutant strains in the ‘unc’ and pseudo wild type groups have been screened for altered susceptibility to Bay d9216. The ‘unc’ group of levamisole resistant mutants includes C. elegans carrying mutations in the L-AChR subunits and ancillary proteins involved in the trafficking and assembly of the putative L-AChR. Of the subunits in this group expression of UNC-38, UNC-29 and UNC-63 are essential for the action of levamisole and are expressed at the body wall to form the putative L-AChR (Fleming et al. 1997; Culetto et al. 2004). The ancillary proteins UNC-74, UNC-50 and RIC-3 are essential for the action of levamisole in C. elegans. ric-3 is essential for the maturation of all nAChRs (Halevi et al. 2002). Unc-74 and unc-50 are essential for the specific assembly and trafficking of L-AChRs. The action of levamisole phenocopies mutations in the levamisole resistance genes and leads to the ‘unc’ phenotype (Boulin et al. 2008). Expression of the five subunits and three ancillary proteins is required for the expression of L-AChRs in Xenopus laevis oocytes. (Boulin et al. 2008).

C. elegans carrying mutations in unc-50 or unc-74 are highly resistant to the effects of levamisole (Lewis et al. 1980). In the absence of UNC-50,

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L-AChRs are incorrectly targeted to the lysosome and are degraded (Boulin et al. 2008).

An additional point of interest derives from the discovery that the new anthelmintic monepantel exerts its action through the nAChR subunit acr-23 (Rufener et al. 2010). Therefore a strain carrying a mutation in acr-23 which confers resistance to monepantel has also been included in the analysis. Finally a selection of cholinergic mutant strains which have no reported resistance to anthelmintic compounds were included in the screen.

The mutant C. elegans strains investigated in this chapter are listed in table 11 along with a summary of proposed expression in C. elegans and a selection of parasitic nematodes. The putative expression patterns of the nicotinic genes in the C. elegans nervous system are shown in figure 56.

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Expression confirmed in Reported Gene Mutant C. elegans A. suum H. contortus resistance unc-74 unc-74(e883)  3 - Levamisole unc-50 unc-50(e306)  3 - Levamisole unc-38(e264) Levamisole unc-38  2 (hca-1)1 unc-38(x20) Levamisole unc-29 unc-29(e193)  2 1 Levamisole unc-63 unc-63(b404)  3 1 Levamisole lev-1 lev-1(e211)   1 Levamisole lev-8 lev-8(ok1519)  - X Levamisole acr-23 acr-23(ok2804)  -  (mptl-1) 5 Monepantel acr-8 acr-8(ok1240)  3 Hco-acr-8) 2 Nicotine acr-12 acr-12(ok367)  - - - acr-16 acr-16(ok789)  4 - -

Table 11: List of C. elegans mutant strains tested for altered susceptibility to tribendimidine, Bay d9216 or amidantel. Also shows where expression has been identified or proposed in parasitic species and altered anthelmintic susceptibility where reported. 1(Neveu et al. 2010), 2(Boulin et al. 2011), 3(Williamson et al. 2009), 4(Wang et al. 2011), 5(Rufener et al. 2009).

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Figure 56: Diagram showing the expression pattern of nicotinic subunits and associated proteins in the nervous system.

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5.2. Methods

5.2.1. Thrashing assays of levamisole resistant mutants.

A subset of the levamisole resistant C. elegans strains has severely impaired motility. The thrashing rate of wild type and levamisole resistant C. elegans was scored after 10 and 60 minutes in M9 buffer supplemented with 0.1% BSA, in the absence of compound. This was done in order to determine which strains could be reproducibly screened for the inhibitory effects of the cholinergic compounds in liquid.

5.2.2. Body bends assays of levamisole resistant mutants.

The number of body bends produced by levamisole resistant worms on agar plates dosed with 100 µM tribendimidine, amidantel, Bay d9216, levamisole or vehicle control was scored.

5.3. Results

5.3.1. Thrashing assays on levamisole resistant strains

Wild type worms produced a mean average of 2 thrashes per second as did the lev-8(ok1519) strain which is a pseudo wild type levamisole resistant mutant. Unc-38(x20) belonging to the ‘unc’ group, and the pseudo wild type lev-1(e211) thrashed at approximately half the rate of wild type worms. The remaining levamisole resistant mutants tested, which were all ‘unc’ strains, thrashed at a quarter of the rate or less of wild type worms with unc-29(e193) and unc-74(e883) having the most compromised motility in liquid compared to wild type worms.

Using the Student’s t-test, no significant difference was revealed between the thrashing rates of wild type worms at the two time points tested. Of the levamisole resistant mutants the thrashing rate of unc- 38(x20), unc-63(b404) and unc-50(e306) was not sustained and a significant difference between the thrashing rate 10 minutes and after 173

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60 minutes in buffer was revealed with these strains. No significant reduction in thrashing was observed in the remaining strains tested after 60 minutes in buffer. The thrashing rates of wild type worms and the levamisole resistant mutants tested in is shown in figure 57.

2.0

1.5

1.0 Thrashes Hz Thrashes

0.5

*** *** ***

0.0

N2 N2

lev-1(e211) lev-1(e211)

unc-38(x20) unc-22(e66) unc-38(x20) unc-22(e66)

unc-38(e264) unc-50(e306) unc-29(e193) unc-74(e883) unc-38(e264) unc-50(e306) unc-29(e193) unc-74(e883)

unc-63(b404) unc-63(b404)

lev-8(ok1519) lev-8(ok1519)

10 minutes 60 minutes

Figure 57: The thrashing rate of wild type and levamisole resistant C. elegans after 10 and 60 minutes in drug-free buffer.

The thrashing rate of wild type and levamisole resistant mutant strains was determined 10 and 60 minutes after transfer to drug-free buffer. Data points are the mean (±se) of n≥8, 4 worms per experiment, ≥ 2 experiments. Significance between the 10 & 60 minute time points for each strain was determined using the Student’s t-test, *** p< 0.001.

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The effects of mutations in auxiliary proteins on drug susceptibility

The rate of body bend generation in the unc-50(e306) strain on vehicle control plates was more variable and significantly impaired relative to wild type worms. The body bend generation of this strain in the absence of drug was similar to that of wild type worms after 2 hours on agar plates dosed with compound. When tested using the Student’s t-test, no significant difference was observed between the number of body bends produced by unc-50(e306) on levamisole or amidantel dosed and vehicle control plates, however on plates dosed with 100 µM tribendimidine or Bay d9216 motility was significantly reduced (figure 58).

The number of body bends produced by wild type and unc-74(e884) worms is shown in figure 59. The rate of body bend generation in unc- 74(e884) worms was significantly lower than that of wild type. 100 µM tribendimidine significantly reduced the number of body bends in this strain when tested using the Student’s t-test. Amidantel, Bay d9216 or levamisole had no significant effect on body bend generation in this strain.

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T rib e n d im id in e A m id a n te l (A ) 1 6 (B ) 1 6

1 4 1 4 1

1 1 2 1 2

i m

m 1 0 1 0

s d

d *** n

n 8 8 **

* y

y 6 6

o B B 4 4

2 2

0 0

l l l l o 4 M o 4 M o 4 M o 4 M tr - tr - tr - tr - n 0 n 0 n 0 n 0 o :1 o :1 o :1 o :1 c 2 c 0 c 2 c 0 2 N 0 5 2 N 0 5 N 5 c N 5 c c n c n n U n U U U

B a y d 9 2 1 6 L e v a m is o le 1 6 (C ) 1 6 (D )

1 4 1 4 1

1 1 2

1 2 -

i m

m 1 0

1 0

d n

n 8 8 e

e ***

y 6 6

d o

o *** B B 4 4

2 ** 2

0 0 l l l l M M o M M o 4 o 4 -4 o -4 tr - tr - tr tr n 0 n 0 n 0 n 0 o :1 o :1 o :1 o :1 c 2 c 0 c 2 c 0 2 N 0 5 2 N 0 5 N 5 c N 5 c c n c n n U n U U U

Figure 58: The number of body bends generated by unc-50(e306) on ‘food-dosed’ NGM Plates.

The number of body bends produced per minute by N2 and unc- 50(e306) after 2 hours at 20 °C on ‘food-dosed’ NGM plates with 100 µM (A) tribendimidine, (B) amidantel, (C) Bay d9216 & (D) levamisole or 1% EtOH. Data points are the mean (±se) of n≥6. Significance tested using Student’s t-test. *** p < 0.001, ** p < 0.01, * p < 0.05.

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T rib e n d im id in e A m id a n te l 1 6 1 6 (A ) (B )

1 4 1 4 1

1 1 2 1 2

i m m 1 0

1 0

s d

d *** n

n 8 8 **

y 6 6

B B 4 * 4

2 2

0 0 l l l l o 4 M o 4 M o 4 M o 4 M tr - tr - tr - tr - n 0 n 0 n 0 n 0 o 1 o 1 o 1 o 1 c : c : c : c : 2 4 2 4 2 4 7 2 4 7 N 7 - N 7 - N c N c c n c n n U n U U U

B a y d 9 2 1 6 L e v a m is o le 1 6 (C ) 1 6 (D )

1 4 1 4 1

1 1 2

1 2 -

i m m 1 0

1 0

d n

n 8 8 e

e ***

y 6 6

d o

o *** B B 4 4

2 2

0 0 l l l l o M o M o 4 M o M -4 -4 r - r 4 tr tr t t - n 0 n 0 n 0 n 0 o 1 o 1 o 1 o 1 : : c : c : c 2 c 4 2 4 2 4 7 2 4 7 N 7 N 7 - N c N c c n c n n U n U U U

Figure 59: A comparison of the number of body bends generated by unc-74(e883) on ‘food-dosed’ NGM Plates.

The number of body bends produced by N2 and unc-74(e883) after 2 hours on 100 µM (A) tribendimidine, (B) amidantel, (C) Bay d9216, or (D) levamisole is shown. Data points are the mean (±se) of n≥6. Significance tested using Student’s t-test *** p < 0.001, ** p < 0.01, * p < 0.05.

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The effects of mutations in levamisole receptor subunits on drug susceptibility unc-38

The effect of compounds on the thrashing rate of two alleles of unc-38, a subunit of the L-AChR, was tested. In the absence of compound unc- 38(e264) produced between 20 and 40 thrashes per minute and this rate was highly variable. When tested using the two-way ANOVA, 10 µM tribendimidine and levamisole had no significant effect on the thrashing rate of this strain over 60 minutes. The effect of 10 µM Bay d9216 was found to be significant using the two-way ANOVA test (figure 60). However, prior to addition of vehicle or compound the average rate of thrashing in vehicle control worms tended to be higher than in the group which was then exposed to Bay d9216 and also the groups used to test the effects of tribendimidine and levamisole. The differences between thrashing rates of vehicle control worms were not significant at t=0. Significant differences between the vehicle control groups were found at t=3 & t=11 (p < 0.05) and t=30 (p <0.01).

The second allele tested was unc-38(x20). In the absence of compound unc-38(x20) produced between 40 and 70 thrashes per minute and this rate was again highly variable. When tested using the two-way ANOVA, 10 µM tribendimidine and levamisole had no significant effect on the thrashing rate of this strain over 60 minutes. The effect of 10 µM Bay d9216 was found to be significant using the two-way ANOVA test (figure 61). Prior to addition of vehicle or compound the average rate of thrashing in vehicle control worms was slightly higher than that of the group which was then exposed to Bay d9216 and also the groups used to test the effects of tribendimidine and levamisole. These differences were not significant at t=0. Significant differences between the vehicle control groups were found at t=7 & t=11 (p < 0.05) and t=30 (p <0.01).

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u n c -3 8 (e 2 6 4 )

T rib e n d im id in e L e v a m is o le (A ) (B )

5 0 -5 5 0 -5 0 .1 % D M S O 1 0 M tr i 0 .1 % D M S O 1 0 M le v

4 0 4 0

1 1

- -

n n

i i m

m 3 0 3 0

s s

e e

h h

s s a

a 2 0 2 0

r r

h h T T *

1 0 1 0

0 0 0 2 4 6 8 1 0 2 0 3 0 4 0 5 0 6 0 0 2 4 6 8 1 0 2 0 3 0 4 0 5 0 6 0 T im e m in T im e m in

B a y d 9 2 1 6 (C )

5 0

-5 0 .1 % D M S O 1 0 M B a y

4 0

- n

i 3 0

s a

r 2 0

h *** T ** *** 1 0 ** * ***

0 0 2 4 6 8 1 0 2 0 3 0 4 0 5 0 6 0 T im e m in

Figure 60: The thrashing rate of unc-38(e264) exposed to tribendimidine, levamisole & Bay d9216.

The thrashing rate of individual worms was counted before adding compound to a final concentration of 10 µM in 0.1% DMSO, the thrash rate was then counted at regular intervals for 60 minutes. Data points are the mean (±se) of n≥19 worms, ≥4 worms per experiment. Unc- 38(e264) worms in 10 µM (A) tribendimidine, (B) levamisole & (C) Bay d9216. Significance tested with two-way ANOVA with Bonferroni post- hoc test *** p < 0.001, ** p < 0.01, * p < 0.05. Points below horizontal line = ***. 179

Michelle Joyner Results 3

unc-38(x20)

(A) Tribendimidine (B) Levamisole

80 80 0.1% DMSO -5 0.1% DMSO 10-5 M tri 10 M lev

60 60

-1 -1

40 40

Thrashes min Thrashes Thrashes min Thrashes 20 20

0 0 0 2 4 6 8 10 20 30 40 50 60 0 2 4 6 8 10 20 30 40 50 60 Time min Time min

80 0.1% DMSO 10-5 M Bay

60 -1

40 *

*** Thrashes min Thrashes ** 20

***

0 0 2 4 6 8 10 20 30 40 50 60 Time min

Figure 61: The thrashing rate unc-38(x20) when exposed to tribendimidine, levamisole or Bay d9216.

Michelle Joyner Results 3

To further investigate the effects of the test compounds and levamisole on unc-38(x20), the number of body bends produced by wild type worms after 2 hours on ‘agar-dosed’ plates with 10 - 100 µM tribendimidine, amidantel, Bay d9216, levamisole or 0.1% vehicle control was scored in wild type and unc-38(x20). The one-way ANOVA test showed the rate of body bend generation was significantly inhibited in wild type worms exposed to each acetanilide compound.

The body bend generation of unc-38(x20) was slightly reduced but not significantly different to that of wild type worms on vehicle control plates. After 2 hours on amidantel plates the rate of body bend generation was significantly inhibited but only at the higher concentration tested. 10 and 100 µM tribendimidine or Bay d9216 significantly inhibited body bend generation in this strain. The observed inhibition was concentration dependent. Levamisole did not inhibit body bend generation (figure 62).

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N2 unc-38(x20)

(A) (B) 14 14

12 12 -1 10 -1 10

8 8 *** *** 6 6

4 Body bends min bends Body

Body bends min bends Body 4

2 2

0 0 Amidantel & tribendimidine & Amidantel

-5 M tri -4 M tri -5 M tri -4 M tri -5 M ami -4 M ami -5 M ami -4 M ami 10 10 10 10 10 10 10 10 0.1 % DMSO 0.1 % DMSO

12 12 -1 10 -1 10

8 *** 8 *** 6 6

Body bends min bends Body 4

2 2

0 0 Levamisole & Bay d9216 Bay & Levamisole

NGM NGM M lev M lev -5 M lev -4 M lev M bay -5 -4 -5 M bay -5 -4 M bay -4 M bay 10 10 10 10 10 10 10 10

Figure 62: The inhibition of body bends in N2 or unc-38(x20) worms on ‘agar-dosed’ Plates.

The number of body bends produced per minute following incubation on ‘agar-dosed’ plates. Data points are the mean (±se) of n≥6. (A) Body bends produced by N2 worms on 10 -100 µM tribendimidine or amidantel plates. (B) Body bends in unc-38(x20) on 10 - 100 µM tribendimidine or amidantel. (C) The number of body bends in N2 on 10 - 100 µM Bay d9216 or levamisole (D) Body bends produced by unc- 38(x20) on 10 - 100 µM Bay d9216 or levamisole. One-way ANOVA with Bonferroni post-hoc test, *** p <0.001 compared to control. All points below horizontal line = ***. 182

Michelle Joyner Results 3 unc-29

UNC-29 is an essential subunit of the putative levamisole receptor (Lewis et al. 1980). Unc-29(e193) was one of the most severely debilitated strains in thrashing in compound free buffer (figure 57). The rate of body bend generation on agar was also greatly reduced relative to wild type worms. When incubated on ‘food-dosed’ plates with 100 µM tribendimidine, amidantel and Bay d9216 for 2 hours the rate of body bend generation in this strain was significantly reduced relative to vehicle control when tested using the Student’s t-test. No reduction in body bend generation was observed in this strain on levamisole plates (figure 63).

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T rib e n d im id in e A m id a n te l 1 6 (A ) 1 6 (B )

1 4 1 4

1 1 2 1

1 2 -

i m

m 1 0

1 0

s d

d *** n

n 8 8 **

y 6

6 d

o B B 4 4 ** 2 2 *

0 0 l l l l o M o M o M o M r -4 r -4 r -4 r -4 t t t t n 0 n 0 n 0 n 0 o 1 o 1 o 1 o 1 c : c : c : c : 2 9 2 9 2 9 2 2 9 2 N 2 N 2 N c N c c n c n n U n U U U

B a y d 9 1 2 6 L e v a m is o le (C ) 1 6 (D ) 1 6

1 4 1 4 1

1 2 1

- 1 2

i m 1 0 m

1 0

d n

8 n 8 e

e ***

6 y 6

d o

*** o B 4 B 4

2 2 ** 0 0

l l l l o 4 M o 4 M o 4 M o 4 M tr - tr - tr - tr - n 0 n 0 n 0 n 0 o 1 o 1 o 1 o 1 : : c : : c 2 c 9 2 c 9 2 9 2 2 9 2 N N 2 N 2 c N c c n c n n U n U U U

Figure 63: The number of body bends generated by unc-29(e193) relative to N2 on ‘food-dosed’ plates.

The number of body bends produced per minute in worms incubated on plates dosed with 100 µM (A) tribendimidine, (B) amidantel, (C) Bay d9216 or (D) levamisole. Data are the mean (±se) of n≥6. Significance tested with Student’s t-test *** p < 0.001, ** p < 0.01, * p < 0.05 compared to 1% control.

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Mutations in the genes expressing LEV-8 and LEV-1 lead to pseudo wild type worms which are partially resistant to the effects of levamisole (Fleming et al. 1997). The lev-8(ok1159) strain thrashed at a similar rate to wild type worms and the thrashing rate was sustained over the 30 minute period these worms were tested. When exposed to 10 µM Bay d9216 or levamisole no significant inhibition was observed when tested using the two-way ANOVA. During the first 5 minutes of exposure to Bay d9216 the rate of thrashing was reduced, but this rate recovered. A similar reduction was seen after 30 minutes exposure to Bay d9216, neither reductions in thrashing was significant. Exposure to 10 µM levamisole also caused a small, insignificant reduction in thrashing after 10 minutes exposure and this rate remained slightly lower than that of control worms for the remaining assay period (figure 64).

Prior to application of compounds the thrashing rate of lev-1(e211) was quite variable, thrashing between 40 and 80 times per minute, the thrashing rate was reasonably stable over the 30 minute assay period. When exposed to 10 µM levamisole a reduction of thrashing rate was observed which was maximal after 10 minutes exposure, the rate of thrashing then returned to that of vehicle control worms. The reduction in thrashing was not significant. When this strain was exposed to 10 µM Bay d9216 a reduction in thrashing rate was immediately observed and remained consistently lower than that of control, however as the thrashing rate of control worms was variable the reduction in thrashing was only found to be significant at the final time point (t=30 min) when tested using the two-way ANOVA (figure 65).

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140 0.1% DMSO 10-5 M Bay d9216 10-5 M levamisole

120

100 -1

60 Thrashes min Thrashes 40

0 0 5 10 15 20 25 30 Time (min)

Figure 64: The effect of Bay d9216 or levamisole on the thrashing rate of lev-8(ok1519).

Data points are the mean (±se) of n≥4. Using the two-way ANOVA with Bonferroni post-hoc test no significant effect was observed with either Bay d9216 or levamisole relative to 0.1% DMSO control.

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0.1% DMSO 10-5 M Bay d9216 10-5 M levamisole 80

60 -1

40 Thrashes min Thrashes 20

* 0 0 5 10 15 20 25 30 Time (min)

Figure 65: The effect of Bay d9216 or levamisole on the thrashing rate of lev-1(e211).

Data points are the mean (±se) of n≥4. Two-way ANOVA with Bonferroni post-hoc test no significant effect with levamisole relative to 0.1% DMSO control, * p < 0.05 at t=30 min with Bay d9216.

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5.3.2. Screen of non-levamisole resistant mutants.

Monepantel (AAD) resistant strain.

The primary target for this type of compound in C. elegans has been identified as being the ACR-23 containing receptor, acr-23 is also a member of the DEG-3 subfamily of nAChRs (Kaminsky et al. 2008).

The rate of thrashing in acr-23(ok2804) was reasonably well sustained during the 30 minute assay period and was comparable to wild type thrashing rates. The thrashing rate at the final time point was slightly reduced relative to previous time points. Exposure to 10 µM Bay d9216 or levamisole caused an initial inhibition of thrashing rate in this strain. The thrashing rate was highly variable for the first 10 minutes of exposure to either compound and the inhibition was insignificant when tested using the two-way ANOVA. The inhibition of levamisole exposed worms was partially reversed after approximately 5 minutes exposure. After 20 minutes exposure to either Bay d9216 or levamisole thrashing was significantly inhibited. An increase in thrashing rate and variability was observed after 30 minutes which meant that inhibition was not significant at this time point (figure 66).

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140 0.1% DMSO 10-5 M Bay d9216 10-5 M levamisole

120

100 -1

60 Thrashes min Thrashes 40

* 20 ** 0 0 5 10 15 20 25 30 Time (min)

Figure 66: The effect of Bay d9216 or levamisole on the thrashing rate of acr-23(ok2804).

Data points are the mean (±se) of n≥4. Significance was tested using the two-way ANOVA with Bonferroni post-hoc test, * p<0.05, ** p <0.01.

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5.3.3. Strains with no reported resistance to current anthelmintics.

The study on levamisole resistant strains described above highlight that Bay d9216 is exerting its inhibitory effects on motility through a set of nAChRs distinct from the L-AChR. Therefore, to establish which subunits are required for Bay d9216 effects a selection of C. elegans strains carrying mutations which have not been implicated in resistance to any of the currently available anthelmintics were screened. The aim of these assays was to reveal if any have altered susceptibility to Bay d9216 relative to wild type worms.

ACR-14 is a non-α subunit which belongs to the ACR-16 like group of C. elegans nicotinic receptor subunits (Mongan et al. 2002). Expression has been confirmed in RME in the head and in ventral nerve cord motor neurons (Fox et al. 2005). In liquid the strain acr-14(ok2804) thrashes at a consistent rate which is similar to wild type worms. When this strain was exposed to 10 µM levamisole the rate of thrashing was significantly inhibited, tested using the two-way ANOVA. Levamisole effects were comparable to wild type. When exposed to the same concentration of Bay d9216 thrashing was significantly inhibited, but the level of inhibition was less than that of wild type worms exposed to 10 µM Bay d9216. Thrashing was quite variable and the rate ranged between approximately 50 and 25% of vehicle control worms (figure 67).

ACR-8 and ACR-12 are both α-subunits and belong to the ACR-8 like group of nAChR subunits (Mongan et al. 2002). ACR-8 is expressed in body wall muscle, and in neurons (Gottschalk et al. 2005). It is levamisole insensitive and nicotine sensitive and is not thought to form part of the functional L-AChR. Its role in the function of body wall muscle is yet to be determined (Brown et al. 2006). Mutations in acr-8 confer resistance to nicotine (Gottschalk et al. 2005). ACR-12 expression is exclusively neuronal, and has been confirmed along with ACR-8 in a

190

Michelle Joyner Results 3 subset of post synaptic clusters which also contain UNC-38 (Gottschalk et al. 2005).

The thrashing rate of strains carrying a mutation in acr-8 or acr-12 is comparable to wild type worms. Thrashing rate was sustained throughout the 60 minute assay period in the absence of compound. Acr-8(ok120) was sensitive to levamisole, exposure to 10 µM levamisole significantly inhibited thrashing when tested using the two-way ANOVA. When this strain was exposed to the same concentration of Bay d9216 no significant inhibition of thrashing was observed. 100 µM Bay d9216 did inhibit thrashing in this strain to a lesser degree and with a slower onset of action than wild type worms. When exposed to 200 µM Bay d9216 thrashing was rapidly and almost completely inhibited for 30 minutes, residual thrashing activity was observed at approximately 10% relative to control worms. At the final time point thrashing was almost completely inhibited in worms exposed to 200 µM Bay d9216 (figure 68).

Acr-12(ok367) was also found to be sensitive to 10 µM levamisole. Thrashing was completely inhibited by this concentration of levamisole within 20 minutes. When the same strain was exposed to 10 µM Bay d9216 thrashing was inhibited by approximately 20% relative to control for 30 minutes, inhibition was not found to be significant when tested using the two-way ANOVA until the final time point at 60 minutes when the rate was just under 50% the thrashing rate of control worms. 100 µM Bay d9216 significantly inhibited thrashing, but again this strain maintained a thrashing rate which was approximately 50% that of control worms for the first 30 minutes and thrashing was not completely inhibited throughout the assay period. Only when acr-12(ok367) was exposed to 200 µM Bay d9216 was thrashing completely inhibited (figure 69).

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0.1% DMSO 10-5 M Bay d9216 10-5 M levamisole 120

100

-1 80

60 ***

Thrashes min Thrashes 40

0 0 5 10 15 20 25 30 Time (min)

Figure 67: The inhibitory effect of Bay d9216 or levamisole on the thrashing rate of acr-14(ok2804).

Data points are the mean (±se) of n≥4. One-way ANOVA with Bonferroni post-hoc test *** p <0.001. All points below horizontal line = ***.

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0.1% DMSO 10-5 M Bay d9216 10-4 M Bay d9216

120 2 x 10-4 M Bay d9216 10-5 M levamisole

100

-1 80

Thrashes min Thrashes 40 * * * * ** ** 20 ***

0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min)

Figure 68: The effect of Bay d9216 or levamisole on the thrashing rate of acr-8(ok1240).

Data points are the mean (±se) of n≥4. Significance tested using the two- way ANOVA with Bonferroni post-hoc test *** p <0.001, ** p < 0.01, * p <0.05. All points below horizontal line = ***.

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0.1% DMSO 10-5 M Bay d9216 10-4 M Bay d9216 140 2 x 10-4 M Bay d9216 10-5 M levamisole

120

100 -1

60 **

Thrashes min Thrashes * 40 ** *** ** *** 20 ***

0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min)

Figure 69: The effect of Bay d9216 or levamisole on the thrashing rate of acr-12(ok367).

Data points are the mean (±se) of n≥4. One-way ANOVA with Bonferroni post-hoc test *** p <0.001 ** p <0.01, * p <0.05 compared to control. All points below horizontal line = *** or ** as specified.

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ACR-16 is an α-subunit of the ACR-16 like group. Expression of this subunit has been confirmed in neurons and the body wall muscle where it forms the proposed homomeric N-AChR (Touroutine et al. 2005). The thrashing rate of acr-16(ok789) was equivalent to that of wild type worms and was sustained throughout the assay period. When exposed to 10 µM levamisole this strain was rapidly and completely inhibited in thrashing as with wild type worms. No inhibitory effect whatsoever was observed when this strain was exposed to 10 µM Bay d9216 (figure 70).

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Michelle Joyner Results 3

0.1% DMSO 10-5 M Bay d9216 10-5 M levamisole 120

100

-1 80

***

Thrashes min Thrashes 40

0 0 5 10 15 20 25 30 Time (min)

Figure 70: The effect of Bay d9216 or levamisole on the thrashing rate of acr-16(ok789).

Data points are the mean (±se) of n≥4. Two-way ANOVA with Bonferroni post-hoc test *** p <0.001. All points below horizontal line = ***.

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Of the strains tested in this screen of cholinergic mutants the strains carrying mutations in acr-12, acr-8 and acr-16 have been found to have reduced susceptibility to Bay d9216 relative to wild type worms while retaining sensitivity to levamisole. A summary of the effects of exposure to 10 µM Bay d9216 in liquid for 30 minutes is shown in figure 71. These strains were selected for an in depth screen for susceptibility to tribendimidine, amidantel, Bay d9216 and levamisole. The inhibitory effects of these compounds in liquid and on agar was tested in experiments paired the inhibitory effects of these compounds on wild type worms.

150

NSD

-1 100 NSD NSD

Thrashes min Thrashes 50

*** 0 - + - + - + - + N2 acr-8 acr-12 acr-16

Figure 71: Summary of the effect of Bay d9216 on the thrashing rate of wild type, acr-8(ok1240), acr-12(ok367) and acr-16(ok789). - = Thrashing rate in M9/BSA, + = thrashing rate in 10 µM Bay d9216. Significance was tested using the Student’s t-test, *** p<0.001, NSD = no significant difference.

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The inhibitory effect of tribendimidine, amidantel, Bay d9216 and levamisole on the locomotion on solid media in wild type and acr- 8(ok1240) worms is shown in figure 72. Generation of body bends in wild type worms exposed to each compound for 2 hours was significantly inhibited relative to vehicle control. Inhibition was dose dependent. Exposure of acr-8(ok1240) worms for the same time period to 10 or 100 µM amidantel or tribendimidine caused a similar dose dependent inhibition of locomotion. When this strain was exposed to levamisole for 2 hours body bend generation was inhibited. Exposure to 10 µM Bay d9216 did not significantly inhibit body bend generation relative to control worms, 100 µM Bay d9216 inhibited body bends, but to a lesser degree than wild type worms tested at the same time. Significance was determined using the one-way ANOVA.

When the same strain was investigated in a thrash assay alongside wild type C. elegans, tribendimidine amidantel Bay d9216 and levamisole significantly inhibited thrashing and inhibition was similar to that observed in wild type worms (figure 73).

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N2 acr-8(ok1240)

(A) (B) 14 14

12 12

-1 -1 10 10

8 8 ** ** * 6 ** 6 *** *** 4 4 ***

Body bends min bends Body *** Body bends min bends Body 2 2

0 0 Amidantel & tribendimidine & Amidantel

-5 M tri -4 M tri -5 M tri -4 M tri -5 M ami-4 M ami -5 M ami-4 M ami 10 10 10 10 10 10 10 10 0.1 % DMSO 0.1 % DMSO

12 12

-1 -1 10 10

8 8 * 6 ** 6 ** ** ***

4 ** 4 ***

Body bends min bends Body Body bends min bends Body 2 2

0 0 Levamisole & Bay d9216 Bay & Levamisole

NGM M lev M lev NGM M lev M lev -5 -4 -5 M bay-4 M bay -5 -4 -5 M bay-4 M bay 10 10 10 10 10 10 10 10

Figure 72: The inhibition of body bend generation in N2 or acr- 8(ok1240) worms on amidantel, tribendimidine, Bay d9216 or levamisole plates. Body bend generation after 2 hours on ‘agar-dosed’ plates. Data points are the mean (±se) of n≥6. Showing the inhibitory effect of 10 -100 µM tribendimidine or amidantel on (A) N2 or (B) acr-8(ok1240), and the inhibitory effect of 10 - 100 µM Bay d9216 or levamisole on (C) N2 or (D) acr-8(ok1240). Significance was compared using the one-way ANOVA with Bonferroni post-hoc test, *** p < 0.001, ** p <0.01, * p < 0.05 compared to control.

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N 2 a c r -8 (o k 1 2 4 0 )

1 2 0 1 2 0 (A ) (B ) 0 .1 % D M S O 1 0 - 5 M trib e n d im id in e

1 0 0 1 0 0 1 0 - 4 M trib e n d im id in e

1 0 - 4 M a m id a n te l

1 -

8 0 - 8 0

s e

6 0 e 6 0

a r

r ***

h h

T 4 0

2 0 2 0 ***

*** *** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

1 2 0 1 2 0 (C ) (D ) B u ffe r 1 0 - 5 M B a y d 9 2 1 6

1 0 0 1 0 0 1 0 - 4 M B a y d 9 2 1 6

1 0 - 5 M le v a m is o le 1

1 - 4 -

- 8 0 8 0 1 0 M le v a m is o le

s e

e 6 0 6 0

s a

a ***

h T T 4 0 4 0

2 0 2 0 ***

*** *** *** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

Figure 73: The inhibition of thrashing in N2 or acr-8(ok1240) worms on amidantel, tribendimidine, Bay d9216 or levamisole. Data points are the mean (±se) of n≥6. Showing the inhibitory effect of 10 -100 µM tribendimidine or amidantel on the thrashing rate of (A) N2 or (B) acr-8(ok1240), and of 10 - 100 µM Bay d9216 or levamisole on (C) N2 or (D) acr-8(ok1240). Significance was compared using the one-way ANOVA with Bonferroni post-hoc test, *** p < 0.001, ** p <0.01, * p < 0.05 compared to control.

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The inhibition of locomotion in acr-12(ok367) was investigated on agar and in liquid. In wild type worms tested in the same experiment each compound significantly inhibited body bend generation after 2 hours on ‘agar-dosed’ plates relative to control worms, inhibition was dose dependent. The rate of body bend generation in acr-12(ok367) was approximately ½ that of wild type worms. Worms incubated for 2 hours on 10 and 100 µM amidantel or levamisole were significantly inhibited, but the inhibition was only dose dependent on amidantel plates. 10 µM tribendimidine or Bay d9216 had no significant effect on the rate of body bends in this strain. The higher concentration of 100 µM tribendimidine had a similar inhibitory effect in acr-12(ok367) to the inhibition in wild type worms. 100 µM Bay d9216 did inhibit body bends in this strain, but to a lesser degree than wild type worms (figure 74).

When tested in a thrashing assay this strain revealed a reduced effect of tribendimidine where only 100 µM caused a significant inhibition and amidantel which had no significant effect on the thrashing rate. Bay d9216 and levamisole inhibited thrashing significantly at both concentrations tested and to the same degree as was observed in wild type worms (figure 75).

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N2 acr-12(ok367)

(A) (B) 14 14

12 12

-1 -1 10 10

8 *** 8

6 6 *

4 4 ***

Body bends min bends Body Body bends min bends Body 2 2 ***

0 0 Amidantel & tribendimidine & Amidantel

-5 M tri -4 M tri -5 M tri -4 M tri -5 M ami -4 M ami -5 M ami -4 M ami 10 10 10 10 10 10 10 10 0.1 % DMSO 0.1 % DMSO

12 12

-1 -1 10 10

8 *** 8

6 6

4 4 ** ** **

Body bends min bends Body Body bends min bends Body 2 2

0 0 Levamisole & Bay d9216 Bay & Levamisole

NGM M lev M lev NGM M lev M lev -5 M bay -4 M bay -5 -4 -5 -4 -5 M bay -4 M bay 10 10 10 10 10 10 10 10

Figure 74: The inhibition of body bend generation in N2 or acr- 12(ok367) worms on amidantel, tribendimidine, Bay d9216 or levamisole plates.

‘agar-dosed’ plates were used to assess the effect of each compound on body bend generation on acr-12-ok367) compared to N2 worms. Data points are the mean (±se) of n≥6. The number of body bends in (A) N2 or (B) acr-12(ok367) on 10 - 100 µM tribendimidine or amidantel. (C) N2 and (D) acr-12(ok367) on 10 - 100 µM Bay d9216 or levamisole. Significance was compared using the one-way ANOVA with Bonferroni post-hoc test *** p <0.001, ** p < 0.01, * p < 0.05 compared to control. All points below horizontal line = ***. 202

Michelle Joyner Results 3

N 2 a c r -1 2 (o k 3 6 7 )

1 4 0 1 4 0 (A ) (B )

1 2 0 1 2 0 0 .1 % D M S O

1 0 - 5 M trib e n d im id in e

1 0 0 1 0 0

1 -

- - 4 n

n 1 0 M trib e n d im id in e

i m

m - 5

8 0 8 0 1 0 M a m id a n te l s

*** s e

e 1 0 - 4 M a m id a n te l

h s

6 0 s 6 0

T T 4 0 4 0

2 0 2 0

*** *** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

1 4 0 (C ) (D ) 1 4 0 1 2 0 B u ffe r 1 2 0 1 0 - 5 M B a y d 9 2 1 6

1 0 0 - 4 1

- 1 0 M B a y d 9 2 1 6

n 1 0 0 - 5 i

1 1 0 M le v a m is o le -

- 4

m i

8 0 1 0 M le v a m is o le

e 8 0

e s

6 0 h

a s

r 6 0

r T *** h

4 0 T 4 0

2 0 2 0 *** *** *** *** *** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

Figure 75: The inhibition of thrashing in N2 or acr-12(ok367) worms in amidantel, tribendimidine, Bay d9216 or levamisole

Individual worms were transferred to buffer and the thrashing rate was determined before, and 30 minutes after adding the compound. The experimenter was blinded as to the treatment. Data points are the mean (±se) of n≥6. The number of thrashes produced by (A) N2 and (B) acr- 12(ok367) worms in 10 - 100 µM tribendimidine or amidantel. (C) N2 and (D) acr-12(ok367) thrash rate in 100 µM Bay d9216 or 10 – 100 µM levamisole. One-way ANOVA with Bonferroni post-hoc test *** p <0.001 compared to control.

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The inhibitory effect of tribendimidine, amidantel, Bay d9216 and levamisole on acr-16(ok789) was investigated over the concentration range 1 – 100 µM. None of the compounds had an inhibitory effect on this strain on plates which were ‘food-dosed’ with 1 µM. Tribendimidine, and amidantel inhibited body bends in acr-16(ok789) after 2 hours on plates dosed with 10 or 100 µM compound and this inhibition was dose dependent. Levamisole caused an inhibition of body bend generation only on plates dosed with 100 µM. No significant inhibition of this strain was observed on plates dosed with 1 – 100 µM Bay d9216 (figure 76). When the inhibitory effects of these compounds on acr-16(ok789) in liquid were investigated 10 – 100 µM amidantel or levamisole inhibited thrashing significantly and similarly to the inhibition observed in wild type worms. The same concentrations of amidantel had a reduced effect relative to wild type worms, no significant effect was observed in acr- 16(ok789) exposed to 100 µM amidantel for 30 minutes. 10 µM amidantel did inhibit thrashing to a lesser degree than that observed in wild type worms. 10 – 100 µM Bay d9216 had no significant effect on the thrashing rate of this strain after 30 minutes (figure 77).

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(A) 13 (B) 13 12 12

11 11 -1 10 -1 10 9 9 8 *** 8 ** 7 *** 7 *** 6 6 5 5

4 4 Body Bends min Bends Body Body Bends min Bends Body 3 3 2 2 1 1

0 0 -6 -5 -4 Control 10-6 10-5 10-4 Control 10 10 10 Log [tribendimidine] M Log [amidantel] M (C) 13 (D) 13 12 12

11 11 -1 -1 10 10 9 9 8 8 7 7 6 6 *** 5 5

4 4 Body bends min bends Body 3 min Bends Body 3 2 2 1 1 0 0 Control 10-6 10-5 10-4 Control 10-6 10-5 10-4 Log [Bay d9216] M Log [levamisole] M

Figure 76: The inhibition of body bend generation by acr-16(ok789) worms on amidantel, tribendimidine, Bay d9216 or levamisole.

The number of body bends per minute was counted after 2 hours on ‘food-dosed’ plates. Data points are the mean (±se) of n≥12, 6 worms per experiment, ≥ 2 experiments. The number of body bends produced by acr-16(ok789) incubated on 1-100 µM (A) tribendimidine, (B) amidantel (C) Bay d9216 or (D) levamisole. Significance was tested with the one-way ANOVA with Bonferroni post-hoc test *** p <0.001, ** p < 0.01 compared to control.

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N 2 a c r -1 6 (o k 7 8 9 )

1 2 0 1 2 0 (A ) (B ) 0 .1 % D M S O 1 0 - 5 M trib e n d im id in e - 4 1 0 0 1 0 0 1 0 M trib e n d im id in e 1 0 - 5 M a m id a n te l

- 4 1

1 * 1 0 M a m id a n te l -

- 8 0 8 0

s e

e 6 0 6 0 h

h ***

s a

a ***

h T T 4 0 4 0 ***

2 0 2 0

*** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

1 2 0 1 2 0 (C ) (D ) B u ffe r 1 0 - 5 M B a y d 9 2 1 6 - 4 1 0 0 1 0 0 1 0 M B a y d 9 2 1 6 1 0 - 5 M le v a m is o le

- 4 1

1 1 0 M le v a m is o le -

- 8 0 8 0

s e

e 6 0 6 0

h s

s ***

h T T 4 0 4 0

2 0 2 0

*** *** *** *** *** 0 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0 0 3 0

T im e (m in ) T im e (m in )

Figure 77: The inhibition of thrashing in N2 or acr-16(ok789) worms in amidantel, tribendimidine, Bay d9216 or levamisole

The thrashing rate determined before, and 30 minutes after adding the compound. The experimenter was blinded as to the treatment. Data points are the mean (±se) of n≥6. The number of thrashes produced by (A) N2 and (B) acr-16(ok789) worms in 10 - 100 µM tribendimidine or amidantel. (C) N2 and (D) acr-16(ok789) thrash rate in 10 -100 µM Bay d9216 or levamisole. Significance was tested using the one-way ANOVA with Bonferroni post-hoc test *** p <0.001, * p < 0.05 compared to control.

The inhibitory effects of the acetanilide compounds and levamisole in each of the mutant strains tested are summarised in tables 12 and 13.

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Tribendimidine Amidantel Bay d9216 Levamisole

Lev resistant Thrash Body bends Body bends Thrash Body bends Thrash Body bends mutant 10 100 100 100 100 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM µM µM µM µM µM

unc-74(e883) ND ND x ND x ND ND x ND ND x

unc-50(e306) ND ND x ND x ND ND x ND ND x

unc-38(e264) x ND ND ND ND  ND ND x ND ND

unc-38(x20) x   x     x x x

unc-29(e193) x ND x ND x ND ND x x ND x

lev-1(e211) ND ND ND ND ND  ND ND x ND ND

lev-8(ok1519) x ND ND ND ND x ND ND x ND ND

Table 12: Summary table showing the effects of tribendimidine, amidantel, Bay d9216 and levamisole on the levamisole resistant strains tested ( = susceptible, X = resistant, ND = not determined).

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Tribendimidine Amidantel Bay d9216 Levamisole Body Body Body Body Thrash Thrash Thrash Thrash bends bends bends bends Mutant 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM

acr-23(ok2804) ND ND ND ND ND ND ND ND  ND ND ND  ND ND ND

acr-8(ok1240)     x      x     

acr-12(ok367) x  x  x      x     

acr-16(ok789)         x x x x   x 

Table 13: Summary table showing the effects of tribendimidine, amidantel, Bay d9216 and levamisole on the non- levamisole resistant strains tested ( = susceptible, X = resistant, ND = not determined).

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5.4. Discussion.

A screen of cholinergic mutant C. elegans strains has been carried out in order to reveal whether any of the compounds tested have the potential to break levamisole resistance. This screen was also undertaken to determine the possible molecular mode of action of amidantel, Bay d9216, or tribendimidine.

In wild type worms the predominant mode of action of all the test compounds has been shown to be an inhibition of locomotion as shown in chapter 3 and this inhibition of locomotion has been utilised to screen a selection of mutant strains. The impaired motility of some levamisole resistant strains has meant that it has been difficult to discern the inhibitory effects of the compound from the inherent effects of mutations which lead to diminished body wall muscle responses. This was particularly the case for some of the ‘unc’ mutants tested. For example the strain unc-50(e306) thrashed at a rate which was less than a quarter that of wild type worms and was unable to sustain thrashing over a 60 minute period. When this strain was tested on agar it has been shown that this strain produced body bends at a similar rate to wild type worms which had been exposed to 100 µM of compound. No significant effect was revealed with any of the test compounds on this strain but exposure to Bay d9216 did reduce the number of body bends relative to vehicle control worms and this tended towards significance. The locomotion of these ‘unc’ strains was often highly variable which led to differences between test groups and had the potential to confound observations. This was seen with the unc-38 strains where differences in the thrashing rates between the control groups were observed. When Bay d9216 effects were tested using these strains a significant effect on thrashing behaviour was revealed but it is not clear whether this was due to a higher thrash rate of the vehicle control group relative to the control group used to test the effects of levamisole. The pseudo wild type strains carrying mutations in lev-1 and lev-8 were highly variable in 209

Michelle Joyner Results 3 thrashing within each group and this variability meant that differences were not found to be significant. Interestingly levamisole had some effect on these strains, particularly lev-1(e211) where an apparent inhibition was observed followed by a reversal of this effect. The initial levamisole effect on this strain appears to be very similar to reported effects on other partially resistant strains. The pseudo wild type strains are reported to be partially resistant to the effects of levamisole as are the ‘twitcher’ group. Mutants in the ‘twitcher’ group are reported to contract initially when placed on 1 mM levamisole plates, this effect is then reversed (Lewis et al. 1980).

It is problematic to draw any firm conclusions from the results of locomotion assays with ‘unc’ strains of levamisole resistant mutants. Their motility is severely compromised and highly variable which makes it difficult to discern the effects of the compound from the effects of the mutation. Nevertheless the data in this section does suggest that Bay d9216 has an inhibitory effect on some of the levamisole resistant strains so hints at an alternative mode of action to levamisole.

The monepantel resistant strain has been shown to be susceptible to both levamisole and Bay d9216. These results show that the ACR-23 receptor is not the molecular target of either Bay d9216 or levamisole.

Strains carrying mutations in acr-8, acr-12 and acr-16 have been shown to be susceptible to the inhibitory effects of levamisole in thrashing where this compound had an equivalent effect as was observed in wild type worms. These strains have been shown to be up to 20 times less sensitive to Bay d9216 relative to wild type worms. These results show that the molecular target(s) of Bay d9216 are distinct from those of levamisole. They also show that Bay d9216 is likely to have more than one molecular target. The strain carrying a mutation in acr-16 is most

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Michelle Joyner Results 3 strongly resistant to the effects of Bay d9216 in thrashing assays, with 10 µM having no inhibitory effect whatsoever relative to control worms. Further tests where the effects of all test compounds in these mutant strains were compared to the effects on wild type worms in both liquid and on agar served to further confirm the reduced activity of Bay d91216 relative to wild type effects and those of levamisole. Acr- 8(ok1280) was found to have a reduced response to amidantel in thrashing inhibition, as was acr-12(ok367), which also had a reduced response to 10 µM tribendimidine. In further tests with this strain Bay d9216 did have an inhibitory effect suggesting that if this is a target for the molecular mode of action for Bay d9216 it is not an essential target. Acr-16(ok789) showed the highest level of resistance to Bay d9216 in both thrashing and body bend assays, results which strongly suggest that this is an essential target for the actions of Bay d9216. ACR-8 is reported to be expressed in body wall muscle but its role has not been clearly defined. ACR-12 is a neuronally expressed subunit which has been associated with the levamisole sensitive UNC-38 subunit. ACR-8 and ACR-12 may be implicated in general neuromuscular signalling therefore targeted by both levamisole and Bay d9216 (and other cholinergic agonists).

5.5. Summary

The reverse genetic screen highlighted levamisole-resistant breaking effects of Bay d9216 and a specific role for ACR-16 in its inhibitory effect on motility. ACR-16 is reported to be the essential receptor in the nicotine sensitive and levamisole insensitive receptor located at the body wall muscle (Touroutine et al. 2005), it is also likely to be involved in the regulation of egg laying in hermaphrodite C. elegans (Kim et al. 2001). Receptors containing this subunit are not implicated in the action of levamisole or related compounds so if this is the main target for the molecular mode of action of Bay d9216, this compound does have the

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Michelle Joyner Results 3 potential to break levamisole resistance through a distinct molecular mode of action. The next chapter provides an in depth analysis of the role of ACR-16 in the mode of action of Bay d9216.

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Chapter 6. The selective interaction of Bay d9216 with ACR-16

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6.1. Introduction

In the previous chapter a strain carrying a null mutation in ACR-16 was shown to be highly resistant to Bay d9216 suggesting this compound may be a ligand for the N-AChRs on body wall muscle which contain the ACR-16 subunit. In this chapter this observation has been extended to i) explore whether or not acr-16 is required for the mode of action of any of the other compounds under investigation ii) validate the resistance of acr-16 to Bay d9216 in further behavioural assays i.e. body bends and progeny number iii) a full time-course for the thrashing assay with acr-16 and iv) to directly compare the effects of Bay d9216 and monepantel in acr-16 and acr-23 mutants.

6.2. Methods

Behavioural assays were performed as previously described in chapter 3 and 4.

6.3. Results

In acr-16(ok789) amidantel, tribendimidine and levamisole all inhibited body bend generation relative to control worms and this inhibition was greater than that observed in wild type worms. Bay d9216 had no significant effect on body bend generation in acr-16(ok789). A small but insignificant reduction of body bends was observed in this strain as with previous investigations (figure 78). Further investigations into the body bend generation of acr-16(ok789) on agar plates dosed with 1 – 100 µM Bay d9216 were undertaken and paired with the effects of this compound on wild type worms. Using the one-way ANOVA test a significant and dose dependent inhibition of body bends relative to vehicle control was observed in wild type worms at all doses tested. This was comparable to previous tests. When acr-

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16(ok789) was tested with the same concentration range no significant inhibition was observed in body bend generation. As with all previous tests, a small but insignificant reduction in body bend rate was observed which was not concentration dependent (figure 79).

N2 acr-16(ok789)

(A) 14 (B) 14

12 12

-1 -1 10 10 8 8 ** 6 ** 6 ***

4 4 *** Body bends min bends Body Body bends min Body bends 2 2 0 0

-5 M tri -5 M tri -5 M ami -5 M ami

amidantel & tribendimidine & amidantel 10 10 10 10 0.1 % DMSO 0.1 % DMSO

12 12

-1 -1 10 10 8 8 6 ** 6 ***

4 4 *** Body bends min bends Body Body bends min bends Body 2 2 0 0

-5 M lev -5 M lev levamisole & Bay d9216 & Bay levamisole -5 M bay -5 M bay NGM only 10 NGM only 10 10 10

Figure 78: The number of body bends generated by N2 or acr-16(ok789) on ‘agar-dosed’ Plates.

Compound was added to liquid agar and this was used to pour test plates. After 2 hours at 20 °C the number of body bends produced per minute was scored. Data points are the mean (±se) of n≥6. 10 µM tribendimidine or amidantel inhibited body bends in (A) N2 worms and (B) acr-16(ok789) worms. The effect of 10 µM Bay d9216 or levamisole (C) N2 or (D) acr-16(ok789) worms is shown. Significance tested using the One-way ANOVA with Bonferroni post-hoc test * p < 0.05, *** p <0.001 relative to control.

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(A) 12 (B) 12 11 11

10 ** 10 -1 -1 9 9 *** 8 8 7 7 6 *** 6 5 5

4 4 Body bends min bends Body Body bends min bends Body 3 3 2 2 1 1 0 0 Control 10-6 10-5 10-4 Control 10-6 10-5 10-4 Log [Bay d9126] M Log [Bay d9126] M

Figure 79: A comparison of the effects of Bay d9216 on wild type and acr-16(ok789) on ‘food-dosed’ NGM plates.

The number of body bends produced by (A) N2 worms and (B) acr- 16(ok789) worms on plates food-dosed with 1 – 100 µM Bay d9216. Significance was tested with the one-way ANOVA with Bonferroni post- hoc test, *** p <0.001, ** p <0.01. Data points are the mean (±se) of n≥24, ≥ 4 experiments.

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The effects of Bay d9216 on the progeny production of this strain were tested using the same methodology as described with wild type worms in chapter 4. In the absence of compound, acr-16(ok789) produced a similar number of offspring to wild type worms. 50 – 200 µM Bay d9216 had no significant effect on the number of progeny produced by this strain (figure 80).

(A) 200 (B) 200 NSD

150 150

100 100

No. progeny No. No. progeny No. 50 50

0 0 -5 -4 -4 -5 -4 -4

5 x 10 1 x 10 2 x 10 5 x 10 1 x 10 2 x 10 0.1% DMSO 0.1% DMSO [Bayd9126] M [Bayd9126] M

Figure 80: The effect of Bay d9216 on the number of progeny produced by wild type and acr-16(ok789) C. elegans adults. The number of offspring produced by L4 worms after 3.5 days at 20°C in the wells of a 48-well microtitre plate containing 50 – 200 µM Bay d9216 in M9/BSA buffer and E. coli OP50 (OD600=3). (A) Wild type worms (Shown previously (chapter 4, figure 48)) and (B) acr-16(ok789). Data points are the mean brood size (±se) of ≥3 experiments, significance tested using the one-way ANOVA with Bonferroni post-hoc test, ** p < 0.01 compared to vehicle 0.1% DMSO. All points below horizontal line = NSD.

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In experiments shown in the previous chapter in wild type worms, 10 µM tribendimidine, Bay d9216 or levamisole was sufficient to almost completely inhibit thrashing. This concentration of tribendimidine had a significantly inhibitory effect on the thrashing rate of acr-16(ok789) relative to control worms. The observed inhibition was reduced relative to previous tests with wild type worms and some reversal of tribendimidine inhibition was seen between the 30 and 60 minute time points (figure 81, A). In order to determine the time course of the effect of the compound on this mutant strain the effects of 10 µM Bay d9216 on the thrashing rate of acr-16(ok789) was compared to vehicle control worms. This concentration of Bay d9216 caused an initial, small, but significant reduction in thrashing rate in this strain relative to control worms. This effect was rapidly reversed and worms were not significantly inhibited in thrashing within 5 minutes of exposure and for the remainder of the test period relative to control worms (figure 81, B). 10 µM levamisole inhibited the thrashing of this strain in a manner comparable to wild type worms. Inhibition was rapid in onset and worms were completely inhibited within 20 minutes. Complete inhibition remained for the duration of the experiment. These observations were comparable to the effects of this concentration of levamisole in wild type worms (figure 81, C). The experimenter was blinded as to the treatment in half the experiments shown in figure 81.

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(A) 150 0.1% DMSO 10-5 M tribendimidine

125 -1 100 *** 75

50 Thrashes min Thrashes 25

0 0 2 4 6 810 20 30 40 50 60 Time min

(B) 150 0.1% DMSO 10-5 M Bay d9216

125 -1 * 100 ***

50 Thrashes min Thrashes 25

0 0 2 4 6 810 20 30 40 50 60 Time min

125 -1 100 *** 75

50 Thrashes min Thrashes 25

0 0 2 4 6 810 20 30 40 50 60 Time min

Figure 81: The inhibition of thrashing in acr-16(ok789) exposed to 10 µM tribendimidine, Bay d9216 or levamisole in liquid.

The thrashing rate of worms in buffer supplemented with 10 µM (A) tribendimidine, (B) Bay d9216, or (C) levamisole is shown. Data points are the mean (±se) of n≥20 from ≥ 4 experiments. Significance tested with one-way ANOVA with Bonferroni post-hoc test *** p <0.001 relative to control. All points below horizontal line = ***.

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Monepantel is reported to exert its effects through the AChR subunit ACR-23 (Rufener et al. 2010). To determine whether the ACR-16 subunit is involved in the action of monepantel the effects of this compound on the thrashing behaviour of acr-16(ok789) over time was tested. These experiments were paired with the effects of monepantel in wild type and acr-23(ok2804). In each strain tested 100 µM and 1mM monepantel had no inhibitory effect for the first 30 minutes of exposure. Wild type worms were significantly inhibited (when tested using the two-way ANOVA) after 30 minutes exposure to 1 mM monepantel, the lower concentration only inhibited wild type worms after exposure for 1 hour. Inhibition was dose dependent in wild type worms. When tested with acr-16(ok789) monepantel had a slower onset of action relative to wild type worms. When tested using the two-way ANOVA, no significant inhibition of thrashing was observed until this strain had been exposed for 2 hours when a dose dependent inhibition which was comparable to that seen in wild type worms was observed. The monepantel resistant strain, acr-23(ok2804) was found to be completely resistant to the inhibitory effects of monepantel over the concentration range and the time course tested. The effects of monepantel in these strains over time are shown in figure 82. The percentage inhibition of each strain after exposure to 0.1 – 1 mM monepantel relative to 0.1% DMSO control summarised in figure 83.

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(A) N2 120 0.1% DMSO 10-4 M monepantel -3 100 10 M monepantel

-1 80

** 60 ** ** *** *** ***

Thrashesmin 40

0 0 10 20 30 40 50 60 70 80 90 100 110 120 Time min

acr-16(ok789) (B) 120 0.1% DMSO 10-4 M monepantel 100 10-3 M monepantel

-1 80 ** 60

*** Thrashesmin 40

0 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min)

acr-23(ok2804) (C) 120 0.1% DMSO 10-4 M monepantel -3 100 10 M monepantel

-1 80

Thrashesmin 40

0 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min)

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Figure 82: The effect of monepantel on the thrashing rate of N2, acr- 16(ok789) and acr-23(ok2804) worms.

(Previous page). The thrashing rate of (A) N2, (B) acr-16(ok789), and (C) acr-23(ok2804) worms in 0.1 – 1 mM monepantel is shown. Data points are the mean (±se) of n≥6, 6 worms per experiment. Significance was compared with the two-way ANOVA with Bonferroni post-hoc test *** p <0.001, ** p <0.01 compared to control.

150

NS NS 100

** ** *** % Inhibition % *** 50

0 -4 -3 -4 -3 -4 -3 10 10 10 10 10 10 Log [monepantel] M

N2 acr-16(ok789) acr-23(ok2804)

Figure 83: A summary of the inhibition of thrashing in wild type, acr- 16(ok789) & acr-23(ok2804). The inhibition of thrashing is shown as a percentage of control (0.1% DMSO) after exposure for 2 hours in 100 µM – 1 mM monepantel. Significance was tested with the one-way ANOVA with Bonferroni post- hoc test *** p <0.001, ** p <0.01 compared to control.

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6.4. Discussion

In the previous chapter preliminary data were shown which suggested that the primary target for the inhibitory effects of Bay d9216 on locomotion was AChRs comprised of ACR-16. In this chapter further investigations into the selectivity of Bay d9216 have served to validate and reinforce these preliminary data. These investigations have also shown the effects of Bay d9216 are distinct to those of amidantel, tribendimidine and levamisole.

In the investigation of Bay d9216 inhibition of body bends in acr- 16(ok789) worms a small, insignificant reduction in body bend generation was consistently observed which is likely to be due to this compound targeting multiple receptor subunits such as ACR-12 or ACR- 8 which are still present and functional in this mutant strain. As the observed inhibition was very small it is likely that the principal molecular target for Bay d9216 is ACR-16. It would be interesting to test the double mutant acr-16::acr-12 or acr-16::acr-8 or indeed the triple mutant acr-16::acr-12::acr-8.

A similar trend was observed in investigations of the effects of tribendimidine, Bay d9216 and levamisole in thrashing behaviour in acr- 16(ok789). Thrashing in this strain was rapidly and almost completely inhibited when exposed to tribendimidine or levamisole but not Bay d9216. As with body bend generation, a small inhibitory effect was observed which was not sustained. This further suggests that Bay d9216 is multigenic in its action with ACR-16 being the key target.

The recently discovered anthelmintic monepantel was tested in thrashing assays with acr-16(ok789). These investigations were paired with wild type C. elegans and the monepantel resistant strain acr- 23(ok2804). The results of these experiments showed that monepantel has a comparable inhibitory effect in both acr-16(ok789) and wild type

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Michelle Joyner Results 4 worms and confirms that the target of Bay d9216 is not shared with that of monepantel.

Exposure to Bay d9216 did not reduce the number of progeny produced by acr-16(ok789) relative to vehicle controls or wild type worms. This lack of effect further corroborates the evidence presented that ACR-16 is an essential target for the activity of this compound. The absence of functional receptors containing this subunit leads to strong resistance to Bay d9216 in both inhibition of locomotion and in production of progeny. Receptors containing this subunit have not previously been implicated in the activity of any currently available anthelmintic compounds.

6.5. Summary

In conclusion these data highlight a specific role for ACR-16 in the mode of action of Bay d9216. As ACR-16 containing receptors have been shown to contribute to ~85% of the cholinergic current at the body wall muscle (Touroutine et al. 2005), these findings are in agreement with the effects of this compound in wild type worms shown in previous chapters where an inhibition of locomotion was the predominant effect of Bay d9216.

This is an important finding as there are no known anthelmintics which specifically target receptors containing this subunit. These data not only elucidate the principal mode of action of Bay d9216 at the receptor level, they also show that it has a mode of action which is distinct to that of tribendimidine, amidantel or levamisole.

A monepantel resistant strain was shown in the previous chapter to be susceptible to Bay d9216. The inhibitory effect of Bay d9216 on this strain together with the inhibitory effect of monepantel on acr-16 mutants shown in this chapter further underpins the theory that Bay d9216 has the potential to break resistance in parasitic worm infections.

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Important questions still remain, such as where the ACR-16 receptors required for the action of Bay d9216 are located and whether this compound is acting on receptors located at the nematode body wall muscle. Bay d9216 has been clearly shown to be effective in the model organism C. elegans, it is important to determine whether this compound is effective in parasitic species of nematode. These questions will be approached in the next chapter.

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Michelle Joyner Results 5 Chapter 7. The role of nematode body wall muscle N-AChRs in the mode of

action of Bay d9216

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7.1. Introduction

The reverse genetic screens have provided evidence for a major role of ACR-16 in the mode of action Bay d9216. Importantly, Bay d9216 is effective therefore against C. elegans that are resistant to levamisole and monepantel raising the possibility that it may provide a useful new route to the control of parasitic nematodes.

In this chapter these questions have been probed using a classic rescue approach. This involved the expression of wild-type copies of acr-16 under a body wall muscle specific promoter (myo-3) in acr-16(ok789) to determine whether this restores susceptibility to Bay d9216.

Secondly in this chapter an investigation into whether Bay d9216 has effects in parasitic nematodes was undertaken in order to confirm its potential as an anthelmintic compound. We also aimed to provide evidence of Bay d9216 effects in parasitic worms that were distinct from the effects of levamisole as has been demonstrated in C. elegans.

7.2. Methods

Cloning acr-16 into expression vector

The genomic sequence of acr-16 as reported on the + strand is shown in figure 84. Acr-16 is located on chromosome V, position: 8561247- 8564051. PCR was used to verify the deletion in acr-16(ok789) using 3 different primers, a pair of external primers forward 1 (F1) and reverse (R) and a forward nested primer (F2). The external primer F1 was paired with primer R to amplify a 468 bp fragment from genomic DNA isolated from a developmentally mixed population of acr-16(ok789) worms. The nested primer F2 was paired with R to confirm the deletion in acr- 16(ok789). No fragment was amplified from genomic DNA isolated from a developmentally mixed population or a single acr-16(ok789) worm. To

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Michelle Joyner Results 5 verify activity of the F2 and R primer pair, a 628 bp fragment was amplified from genomic DNA isolated from a population and a single wild type worm (figure 85).

ataaacctta aatattaaat caatgctttt ctttccctga ttctcatgtc cgtATGTCTG TCTGCACCCT TCTCATCTCG TGCGCAATTC TTGCGGCACC GACTCTCGGA TCACTGCAGG AGCGCAGATT GTATGAGGAT TTGATGAGAA ATTATAACAA TCTGGAACGT CCTGTTGCAA ATCATTCCGA GCCAGTTACA GTACATCTAA AGgtattttt aaatttattt ttgatctaaa aatacgatga atgtatgttt tcgaaccatt tgaaaattct tccaaaaacc agagttcgtt ttttgctggt aaatttctag atgaatttca ttttttgtct tctgaagata ttccaaatac agtaataaag ttatgtctac tagaacaatt tgatatttta tttcaaaacg gttactgtat ctaaataata gtaattgtaa ttaatttttt gaaaaaatta atgtaaattt aacttcagGT AGCCCTCCAA CAAATAATTG ACGTAGACGA GAAAAATCAA GTAGTTTATG TAAATGCATG GCTGGATTAT gtaagttttt ggtactttat ttaaaaaaaa aattaaattt catgtattcc agACATGGAA CGACTATAAT TTGGTTTGGG ATAAAGCTGA ATACGGTAAC

ATCACAGATG TCCGTTTTCC AGCTGGAAAG ATCTGGAAAC CAGATGTTCT ATTATATAAC AGTGTTGACA CAAATTTTGA TTCAACGTAT CAAACCAATA TGATTGTGTA TTCAACTGGC TTGGTGCATT GGGTTCCACC GGGAATATTT AAGATTTCAT GTAAAATTGA TATTCAGTGG TTTCCATTTG ACGAGCAAAA ATGTTTCTTT AAAgtaagtt tttactgttt tttttttaaa tttaaatgag aaataattaa tttaaaaatt tttcagTTTG GTTCATGGAC TTATGACGGT TATAAACTTG ATCTTCAACC AGCAACGGGT GGATTTGATA TCAGTGAATA TATTTCAAAC GGAGAATGGG CTTTACCTTg taagttgaaa gatattgaaa aatcaaaatg aactctttct tattaagttt ttgcccaact ttttacccac ttctggaaaa aaaatttcag gaagtttttc attttctacc tgaaatctag atctatagct tttaaagtaa actataaatt atttgtttta ttcaacgttt ttgacacatg tgggaaagtt tacagTGACA ACTGTGGAGC GAAACGAAAA GTTTTATGAT TGCTGTCCGG AACCTTATCC AGATGTTCAT TTTTATCTTC ACATGAGACG GCGAACTCTT TATTACGgta tgttttattt aaatatcatt tgaaggagtt caatttttag GGTTCAACTT AATTATGCCA TGTATATTGA CAACTCTTAT GACACTTCTA GGATTCACAC TTCCTCCTGA TGCAGGAGAG AAAATCACTC TTCgtaagtg aattcttgaa tttgattttt attaaaaaca tattaaactt cagAAATCAC GGTCTTACTT TCAATTTGCT TCTTTTTGAG TATTGTTTCG GAGATGTCAC CTCCAACATC GGAAGCTGTT CCTTTACTAG gtaattctgg tgaattgaat aaatataatg aagttatcaa attgtgcagG TATATTTTTT ACGTGTTGTA TGATTGTGGT TACTGCATCG ACAGTCTTCA CCGTCTACGT 229

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TCTCAACTTA CATTACCGTA CTCCGGAGAC CCACGATATG GGACCATGGg ttagtttttc agaagattgt accatataat ctaaagagtg gattactgtc aataaagacc atgaggaaga tctgaaaata ggatcttttc gtatttctag attctttcat attctaacgc gacagacagt gagtaaaaca aatttagaca ataatctgaa aagtggcaga tcacattcaa aataaactta ttcagACACG TAACCTCCTT CTCTATTGGA TTCCATGGAT TCTTCGAATG AAACGACCCG GTCACAACTT GACATATGCT TCACTTCCAT CATTATTTTC CACTAAGCCA AATCGTCACT CGGAATCATT GATCCGTAAC ATCAAAGACA ATGAACATTC ACTTTCACGA GCAAACTCAT TTGATGCCGA TTGTCGATTG AATCAATATA TTATGACACA ATCTGTTAGT AATGGGTTGA CAAGTCTTGG CAGTATTCCA AGTACAATGA TTTCATCAAA TGGTACAACT ACAGACGTCT CACAACAGGC GACACTTCTG ATTCTTCACA GAATATACCA TGAATTGAAG ATTGTTACGA AGAGAATGAT AGAAGGTGAT AAGGAAGAAC AGGCATGCAA TAATTGGAAA TTTGCGGCCA TGgtacgtac atatatttct cggaagattg aaaattttta agacataagt cctattatta cccagacgta acattttgcg aattctgcga caaagatacg gtaaccggtc tcgacatgac aatttttttg aaatataaaa acactgcgcg cctttaaata ttactgtaat ttcaaacttt cgttgctgca aaattttcat cgagttttca ttgttttcta tgaaaattta tatttgaaaa ctaaccttta aaggcgcaca tccgcttgta tttaatgaaa aattttcgcg tcgagatcgg gaaccgtatt cttgacgcaa atattgcaaa agctcgcacc tcagtaatag tgctattagt ttgaaaaaat ttaaaataaa actagaaact tttagGTTGT GGACCGCCTT TGTTTATACG TCTTCACAAT ATTCATAATT GTTTCAACGA TTGGAATTTT CTGGTCAGCA CCGTATCTTG TCGCCTAAtg atgaaagttt acttgtaatt aatatttaat caactgttct tcttcttctt tgtttttttt tctttgtaat attcattgct ttcgagcttt caaatagtca ttttgttcct ttcgttccat ctgaattgtg atttttgaca catcttcttc gctgtcctcc tcctctttga ttagtctttt caatcttcgc ttctatgccg acgtatttat atcgttcgaa aagaaacttt tttgatcact gtgat

Figure 84: The genomic sequence of acr-16. The primers used to verify the deletion in acr-16(ok789) are shown as bold text and boxed. Lower case lettering represents introns, upper case denotes exons. Underlined text shows the deleted DNA sequence in acr- 16(ok789). Primer binding sites: F1 forward primer - TCACAGATG TCCGTTTTCC A F2 nested forward primer CAACATC GGAAGCTGTT CCT R reverse primer - ATGGGTTGA CAAGTCTTGG C. 230

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Figure 85: Confirmation of deletion in by PCR on genomic DNA isolated from acr-16(ok789). (A) The genomic location of acr-16 showing the location of the deletion in acr-16(ok789) relative to the wild type gene. (B) The deletion in acr-16 was verified by PCR using F1 forward and R reverse external primers to amplify a 468 bp fragment from genomic DNA isolated from a developmentally mixed population of acr-16(ok789) worms. (C) The deletion in acr-16(ok789) was confirmed by PCR using the nested F2 forward and R reverse primer. Genomic DNA was isolated from a developmentally mixed population (left) and a single L4 worm (right). No amplification was seen with acr-16(ok789) Genomic DNA. A 628 bp fragment was amplified with N2.

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Transgenics

Transgenic strains were generated by microinjection of a wild type copy of acr-16 under the body wall specific promoter myo-3 into the mutant strain acr-16(ok789). The pharyngeal muscle specific transformation marker myo-2::gfp was also injected to allow selection of transformed worms which expressed GFP in the pharynx. This promoter was used as Bay d9216 has been shown to have no effect on the rate of pharyngeal pumping therefore expression of GFP in the pharynx was unlikely to interfere with the effects of the compound. The transgenic strain generated was acr-16(ok789);Ex[P-myo-3::acr-16(+)];Ex[myo-2::gfp]. As a further control to confirm the lack of effect of myo-2::GPF a second strain acr-16(ok789);Ex[myo-2::gfp] was generated to express myo- 2::GFP while still carrying a mutation in the acr-16 gene. Microinjections were performed by Dr James Dillon.

The effects of 10 – 100 µM Bay d9216 on the thrashing rate of wild type, acr-16(ok789);Ex[myo-2::gfp] and acr-16(ok789);Ex[P-myo-3::acr- 16(+)];Ex[myo-2::gfp] worms was investigated according to protocol described in chapter 3.

Ascaris suum muscle strip preparation

A. suum muscle strips were prepared by cutting a large, healthy, adult worm immediately anterior to the genital pore (a ridge ~ 1/3 down the body). The posterior portion, containing the reproductive tract was disposed of and 1 cm strips were dissected out from the anterior portion. Strips were placed in a 9 cm petri dish containing warmed APF. The muscle strip was obtained by cutting lengthways with a very sharp scalpel along the lateral cords and separating the dorsal and ventral halves. Any intestinal debris was then carefully removed with forceps to leave a preparation containing the muscle field. Using standard sewing thread a loop was threaded through one end of the muscle strip and a length of thread was tied through the opposite end. The loop was used 232

Michelle Joyner Results 5 to secure the muscle strip in a holder in the organ bath containing APF at 37°C. The thread was securely tied to the arm of a 25 g isometric transducer (Pioden controls ltd, UK) before topping up with APF to 75 ml. The Software (Powerlabs Chart reader) was calibrated so all read- outs were in grams. The preparation was then subjected to a 1 g pre- load and the muscle strips were allowed to rest until a steady baseline was obtained (~1 hour). All preparations were kept at 37°C throughout.

Experimental Protocol

Muscle strips were prepared as described. Compounds used were dissolved in APF and added to 75 ml organ baths in a 1:100 dilution (1% bath volume). Compounds were washed out, using APF, by at least twice the bath volume. Muscle strips were allowed to return to a steady baseline before addition of further compound.

Haemonchus contortus motility assay

Milton sterilising fluid (Milton) was diluted 1:20 (v/v) in dH O. 100 µl 2 each strain was pipetted into 900 µl Milton solution to ensheathed the larvae. After 30 minutes at room temperature the worm suspension was diluted 1:1 with M9 supplemented with 2% BSA warmed to 37°C. 100 µl of this suspension was added to each of the wells of a 24 well plate prepared with 800 µl M9/2% BSA warmed to 37°C.

The number of motile larvae was scored at 0 hours and any immotile larvae removed. Compounds were dissolved in M9 and warmed to 37°C before pipetting 100 µl compound or buffer into each well. The final concentration of drug wells was 10 or 100 µM. Each condition was replicated 3 times. Motile larvae were then counted at 24, 48 & 72 hours.

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7.3. Results

7.3.1. The effects of Bay d9216 in wild type, acr-16(+), and acr-16(-) C. elegans

Wild type worms were significantly inhibited in thrashing after 30 minutes exposure to 10 – 100 µM Bay d9216 relative to pre-treatment thrash rate when tested using the one-way ANOVA. The control strain acr-16(ok789);Ex[myo-2::gfp] was not significantly inhibited by the same concentration range of Bay d9216. The rescue strain carrying wild type copies of acr-16, acr-16(ok789);Ex[P-myo-3::acr-16(+)];Ex[myo-2::gfp], was significantly inhibited in thrashing relative to the pre-treatment rate when tested using the one-way ANOVA. Inhibition was dose dependent and comparable to that seen with wild type worms (figure 86).

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N2 myo-2::gfp;acr-16(-) (A) (B) 120 120 NS NS

100 100

-1 -1 80 80

60 60

40 40

Thrashes min Thrashes Thrashes min Thrashes 20 20 *** *** 0 0 0 30 0 30 0 30 0 30 0 30 0 30 Time (min) Time (min)

(+) (C) myo-2::gfp;acr-16 120 Buffer 10-5 M Bay d9216 100

10-4 M Bay d9216 -1 80

40 Thrashes min Thrashes 20 *** *** 0 0 30 0 30 0 30 Time (min)

Figure 86: The inhibitory effect of Bay d9216 on the thrashing rate of acr-16(ok789) carrying a wild type copy of acr-16 under the body wall muscle specific promoter myo-3. Thrashing rate was determined before and 30 minutes after adding Bay d9216. Data points are the mean (±se) of n≥18, ≥ 3 experiments. The effect of 10 – 100 µM Bay d9216 on the thrashing rate of (A) N2, (B) acr- 16;Ex[myo-2::gfp] and (C) acr-16(ok789);Ex[P-myo-3::acr-16(+)];Ex[myo- 2::gfp] worms is shown. Significance was tested with the one-way ANOVA with Bonferroni post-hoc test *** p <0.001 compared to control.

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7.3.2. The effects of Bay d9216 in A. suum

The addition of Bay d9216, levamisole or nicotine to A. suum muscle strip preparations caused a dose dependent contraction. Compound was washed from the organ bath at least twice and the muscle was allowed return to baseline levels before adding the next concentration of compound. Addition of increasing concentrations of either Bay d9216 or levamisole produced a bell shaped curve with maximal contraction after application of 10 µM compound. A reduction in the amplitude of contraction was observed when muscle was exposed to 100 µM compound relative to the previous concentration. Application of nicotine led to a curve which was more sigmoidal in shape. Nicotine was less potent than Bay d9216 or levamisole, a final concentration of 100 µM was required to elicit a maximal contraction. Contractile responses were normalised to the maximal contraction elicited with each compound (figure 87).

Cumulative dose response curves were obtained by addition of increasing concentrations of Bay d9216 or levamisole without washing compound out or allowing relaxation of the muscle preparation between applications. Cumulative applications Bay d9216 appeared to be slightly more potent than levamisole in the amplitude of contraction, however when these data were normalised the effects of Bay d9216 and levamisole were indistinguishable (figure 88).

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D o s e re s p o n s e

(A ) 6 B a y d 9 2 1 6 L e v a m is o le N ic o tin e

o i

t 4

o c

t i

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m A

0 -7 -6 -5 -4 -3

L o g c o n c e n tra tio n M

N o rm a lis e d d o s e re s p o n s e

(B ) 1 .5 B a y d 9 2 1 6 L e v a m is o le N ic o tin e

1 0  M = 1 1 0  M = 1 1 0 0  M = 1

o i

t 1 .0

a m

r 0 .5

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L o g c o n c e n tra tio n M

Figure 87: Contraction of A. suum muscle strips in response to Bay d9216, levamisole and nicotine. (A) Dose response curves showing the contractile response in A. suum muscle strips in response to Bay d9216, levamisole and nicotine, n≥3 experiments. (B) Dose response curves normalised to the maximal contractile response which was 10 µM for Bay d9216 and levamisole, 100 µM for nicotine, n≥3 experiments.

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C u m u la tiv e d o s e re s p o n s e (A ) 3 .0 B a y d 9 2 1 6 L e v a m is o le

2 .5

o i

t 2 .0

o c

1 .5

t i

l 1 .0

m A

0 .5

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L o g c o n c e n tra tio n M

N o rm a lis e d C u m u la tiv e d o s e re s p o n s e (B )

1 .5 B a y d 9 2 1 6 L e v a m is o le

o i

t 1 .0

a m

r 0 .5

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Figure 88: Cumulative contraction of A. suum muscle strips in response to Bay d9216 and levamisole. (A) Dose response curves showing the contractile response in A. suum muscle strips in response to cumulative doses of Bay d9216 or levamisole, n≥3 experiments. (B) Dose response curves normalised to 10 µM, n≥3 experiments.

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7.3.3. The effects of Bay d9216 in H. contortus

The effects of Bay d9216 and levamisole on the motility of wild type and multi drug resistant H. contortus larvae were tested. The drug resistant H. contortus used was the White River strain which is an isolate from South Africa which is reported to be resistant to ivermectin and the benzimidazoles (Le Jambre et al. 1995; Riou et al. 2003).

L3 larvae were unsheathed using Milton’s fluid and added to M9 buffer supplemented with 2% BSA. Any immotile larvae were removed and the numbers of motile larvae in each well was recorded before supplementing with Bay d 9216 or levamisole to a final concentration range of 10 – 100 µM. The number of motile larvae was scored after 24 and 48 hours at 37°C.

Wild type H. contortus in the absence of any compound were motile for the 48 hour period tested. All larvae exposed to Bay d9216 or levamisole were significantly inhibited in motility after 24 hours exposure when tested using the one–way ANOVA. 10 – 100 µM Bay d9216 or 10 µM levamisole almost completely inhibited any motility, levamisole was slightly less potent and approximately 1/3 of the larvae exposed to 10 µM levamisole retained some motility after 24 hours. After 48 hours exposure almost all the larvae exposed to 10 – 100 µM Bay d9216 or levamisole were immotile.

The White river strain was approximately 50% less motile than the wild type larvae and motility was less well sustained relative to wild type worms over the same period. Bay d9216 had a reduced effect compared to levamisole in this strain. 10 µM Bay d9216 had no significant effect on motility relative to control worms when tested using the two-way ANOVA. 100 µM Bay d9216 and 10 – 100 µM levamisole did significantly inhibit motility after exposure for 24 hours, albeit to a lesser degree than the effects of these compound in wild type worms. No significant effect was observed with either compound relative to control after 239

Michelle Joyner Results 5 exposure for 48 hours, this is likely to be due to the lack of sustained motility in control worms.

Figure 89: A comparison of the number of motile Haemonchus contortus in buffer supplemented with Bay d9216 or levamisole. The number of motile H. contortus in M9 / 2% BSA was scored before adding 10 – 100 µM Bay d9216 or levamisole. Plates were incubated at 37°C. The number of motile worms at 24 and 48 hours was compared to 0 hours for each condition. (A) Wild type H. contortus. (B) ‘White River’ strain H. contortus. Significance tested using two-way ANOVA with Bonferroni post-hoc test, * p <0.05, ** p <0.01, *** p <0.001 relative to t=0.

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7.4. Discussion

C. elegans acr-16(ok789)

Transcription of acr-16 is reported to produce 2 alternatively spliced mRNAs, variant a is 2196 bp and variant b is 1565 bp in length (Thierry- Mieg and Thierry-Mieg 2006). A deletion of 1082 bp in acr-16(ok789) was identified by the CGC and confirmed in (Touroutine et al. 2005). Here, genomic DNA from acr-16(ok789) was amplified by PCR using both external and nested primers. The fragment amplified with external primers was approximately 468 bp and no fragment was amplified using the nested primers. These results verify the reported deletion in this strain.

Transgenic lines expressing wild type copies of acr-16 under the body wall specific promoter myo-3 were generated and selected by expression of the coinjection marker GFP expressed under the pharyngeal muscle promoter myo-2. Worms which strongly expressed GFP in the pharynx were selected from 3 independent lines. These transgenic lines have been shown to be susceptible to the effects of Bay d9216 in inhibition of thrashing. Susceptibility was at least that of wild type worms tested at the same time. The susceptibility of these lines to Bay d9216 confirms that ACR-16 containing receptors are a major molecular target for the inhibitory effects of this compound.

Parasitic worms

The effects of Bay d9216 were compared to those of levamisole and nicotine in wild type A. suum and to levamisole in wild type and multidrug resistant strains of H. contortus.

All three compounds caused contraction in muscle strips from A. suum. Both Bay d9216 and levamisole produced bell shaped curves with maximal contractions after application of a final concentration of 10 µM. Levamisole has been previously reported to be an open channel blocker 241

Michelle Joyner Results 5 at concentrations above 30 µM in A. suum leading to desensitisation (Robertson and Martin 1993). A similar effect in A. suum muscle is reported with acetylcholine, the endogenous agonist of AChRs (Pennington and Martin 1990). Nicotine produced a sigmoidal shaped curve and was less potent relative to the amplitude of contractions produced by Bay d9216 or levamisole.

In investigations using the parasite H. contortus levamisole and Bay d9216 inhibited motility within 24 hours, 10 µM Bay d9216 was slightly more potent than the same concentration of levamisole. At higher concentrations the effects of these compounds was indistinguishable.

In the ivermectin and benzimidazoles resistant White River II strain of H. contortus both Bay d9216 and levamisole inhibited motility within 24 hours at the highest concentration tested, however the relative potency was reversed. The reversed potency of Bay d9216 and levamisole could be due to differing modes of action or to differential regulation of expression of AChR subtypes in this isolate. Further investigations using a levamisole resistant strain would be required before any firm conclusions can be drawn.

7.5. Summary

The verification of the reported deletion in the acr-16 mutant strain has served to validate previous findings presented here that that the lack of susceptibility to Bay d9216 in this strain was due to the lack of functional N-AChRs. This was further reinforced by the restored susceptibility to Bay d9216 in a transgenic strain expressing wild type copies of acr-16 under a body wall specific promoter in an acr- 16(ok789) background. Together these data corroborate data presented in previous chapters that ACR-16 subunits are the major molecular target for Bay d9216.

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The effects of Bay d9216 in parasitic nematodes in both muscle contraction and motility of the whole worm confirm that Bay d9216 is active in both species tested. Bay d9216 has been shown to be at least as potent as levamisole in wild type A. suum and H. contortus.

These compounds both showed a similar desensitisation effect in A. suum which was different to nicotine where desensitisation was not observed. It would be of interest to investigate the blockade of the effects of Bay d9216 and levamisole using selective antagonists. This should aid in differentiating the effects of these compounds at the receptor level in parasitic nematode species.

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Michelle Joyner Conclusion

Chapter 8. Conclusion

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Human infections with parasitic worms are estimated to affect over 1/3 of the global population, with a disproportionate bias towards those in third world and developing countries. Over 1 billion people in sub- Saharan Africa, Asia, and the Americas are infected with at least one species of parasitic worm (Hotez et al. 2008; Lustigman et al. 2012). Globally, 120 million people are estimated to be infected with lymphatic filariasis, up to 1472 million are infected with ascariasis and up to 1300 million with hookworm infections (Lustigman et al. 2012).

In agriculture anthelmintic resistance is already widespread in small ruminants and horses and is emerging in cattle and pigs (Prichard 1994). Infection with parasitic worms in agriculture has extensive implications including reduced weight and body condition, increased productivity costs and reduced output (Miller et al. 2012). The ever increasing global demand for food, which is estimated to increase by 75% within 40 years (Keating et al. 2010), means that the growing problem of anthelmintic resistance in agriculture is a real threat to future food security.

The increase in parasitic worm infections which are resistant to available anthelmintics is the driving force behind the need for the development of new anthelmintic compounds with novel modes of action. This project had two main aims. The first was to extend on previous investigations into the effects of tribendimidine, amidantel and Bay d9216 in order to determine the predominant effect of these compounds and to compare them with the effects of the widely used cholinergic anthelmintic levamisole. This has been undertaken using C. elegans as a model organism to investigate the effects of these compounds on worm physiology and development.

The second aim has been to determine the molecular mode of action of these compounds. The elucidation of the molecular targets of the acetanilides will not only inform on the mode of action of these compounds and clarify whether they have a resistance breaking 246

Michelle Joyner Conclusion potential but may prove useful in the development of future compounds. It may also identify potential anthelmintic targets by improving understanding of the physiology of nematodes using C. elegans as a model. The use of C. elegans in this project has proved to be a valuable and valid tool in discerning the effects of these compounds.

Previous studies have provided evidence that amidantel and its derivatives act as cholinergic agonists and are likely to act at on nAChRs located at the NMJ (Tomlinson et al. 1985; Hassoni et al. 1988). This project has shown that, of the behaviours tested, an inhibition of locomotion was the predominant effect of all the compounds tested in agreement with the compounds acting as cholinergic agonists at the NMJ. At least three pharmacologically distinct nAChRs have been identified at the nematode NMJ, the monepantel sensitive ACR-23 receptor, the levamisole sensitive L-AChR and the nicotine sensitive N- AChR. Relative to the L-AChR the N-AChR is largely uncharacterised, however recent reports have shown that the putative N-AChR is a homomeric receptor consisting of ACR-16 subunits (Touroutine et al. 2005).

Initial evaluation of different dosing protocols and has shown that dosing in liquid was found to be the optimal protocol to investigate dose dependency, kinetics, reversibility and the relative potency of the test compounds. This method gave the tightest control over both the dosage and the time course of exposure. The addition of drug to agar was used in some assays. This method of dosing the worms has proved useful in egg laying, brood size and development assays where it was important to be able to accurately count the number of eggs or offspring and to allow the development of larvae to be followed. The use of body bend assays on agar was particularly useful where mutant strains were unable to thrash or maintain thrashing in liquid. Compounds applied using the agar based methods were shown to have

247

Michelle Joyner Conclusion an inhibitory effect on the locomotion of wild type worms, however this effect was not as striking as was observed with dosing in liquid both in onset and potency of drug action and the maximum inhibition observed using this method was approximately 50% of control with 100 µM compound compared to 100% of control when liquid dosing was used at the same concentration.

The onset of inhibition and IC values derived from these data were 50 comparable between compounds. All compounds tested were very rapid in their onset of action showing that the nematode cuticle did not represent a major barrier to these compounds which were able to rapidly reach their target. IC values ranged from 1 – 4 µM with a 50 relative order of potency of tribendimidine > levamisole > Bay d9216.

The analysis of reversal of drug effects revealed interesting differences between tribendimidine and Bay d9216. Tribendimidine exposed worms showed no reversal of inhibitory effects in worms previously exposed to >10 µM and this was also shown in levamisole exposed worms. In contrast the effects of 100 – 200 µM Bay d9216 were reversed and worms recovered to approximately 50% of control thrashing rate. Worms exposed to 1µM continued to thrash at ~ 50% of control. This revealed an interesting difference in the effects of Bay d9216 compared to levamisole and tribendimidine which is proposed to act through L-AChRs (Hu et al. 2009). The lack of complete reversal of the effects of 100 µM or greater concentrations of Bay d9216 and the lack of reversal of effects of 1µM suggest this compound may be acting on both high affinity and low affinity receptors and/or binding sites and therefore binding at low affinity receptors/binding sites is reversed while binding remains at high affinity sites.

In other behaviours investigated, further differences between the action of levamisole and Bay d9216 were revealed. 100 µM levamisole inhibited the rate of pharyngeal pumping in exposed worms while the same concentration of Bay d9216 had no effect. 100 µM Bay d9216 had no 248

Michelle Joyner Conclusion effect on the number of progeny produced by worms exposed from the L4 larval stage while both tribendimidine and levamisole caused a significant reduction in progeny at this concentration. Only doses of 200 µM caused a reduction in progeny in all three compounds tested. Both Bay d9216 and levamisole stimulated egg laying but 100 µM levamisole stimulated egg laying to a greater degree than the same concentration of Bay d9216. These differences strongly suggested that Bay d9216 acts through a molecular target distinct to that of levamisole or tribendimidine and therefore warranted further investigation. Hence this compound was the main focus in a screen of C. elegans cholinergic mutants in order to reveal strains with altered susceptibility to this compound.

In the reverse genetic screen the mutant strains acr-16(ok789), acr- 12(ok367) and acr-8(ok120) were shown to have reduced susceptibility to Bay d9216 in both liquid and agar dosed assays. acr-16 is a member of the ACR-16 group of C. elegans nAChRs, the latter two are members of the ACR-8 group. None of these subunits have been implicated in the action of levamisole. acr-12 encodes an α-subunit which co-purifies with UNC-29 and LEV-1 suggesting that the ACR-12 subunit can form receptors with UNC-29 and LEV-1 which are both non α subunits (Gottschalk et al. 2005). ACR- 12::GFP is expressed in ventral nerve motor neurons, particularly the D neurons so may mediate synaptic input to these neurons (Cinar et al. 2005; Gottschalk et al. 2005). According to AceView there is only one splice variant of acr-12 which is expressed at high level (1.8 times the average gene) at all developmental stages, expression in the L3 larvae and adult worm is twice that of L1 – L2 larvae (Thierry-Mieg and Thierry- Mieg 2006). acr-8 also encodes an α-subunit with at least 4 splice variants. This gene is expressed at 1.5 times the average gene throughout development

249

Michelle Joyner Conclusion from L1 to adult. Expression levels are similar at each developmental stage (Thierry-Mieg and Thierry-Mieg 2006). ACR-8 is expressed in body wall muscle, some head and tail neurons and nerve cord synapses, but not in the ventral cord motor neurons (Gottschalk et al. 2005). cMYC- tagged UNC-38 subunits were found to co-localise with HA-tagged ACR-8 and ACR-12 in a subset of postsynaptic receptor clusters, showing that these subunits may contribute to some, but not all levamisole receptors (Gottschalk et al. 2005)

Mutations in acr-8 confer nicotine resistance while sensitivity to levamisole is retained. Mutations in acr-12 do not confer resistance to nicotine or levamisole. Therefore while the expression of these subunits overlaps with known levamisole receptor subunits they do not contribute to the levamisole response at these receptors (Gottschalk et al. 2005) . acr-16 encodes an α-subunit with two splice variants. It is expressed at 1.8 times the average gene and expression at all developmental stages has been confirmed with the highest expression in L1 – L3 larvae (Thierry-Mieg and Thierry-Mieg 2006). ACR-16 is an essential subunit of the nicotine sensitive, levamisole resistant nAChR expressed at the body wall muscle and in some motor neurons including DB (Touroutine et al. 2005). This subunit is a homolog of vertebrate α7 subunits, with 47% identity to the chicken α7 (Ballivet et al. 1996). When expressed in Xenopus oocytes and using voltage-clamp electrophysiology, recombinant α7 and ACR-16 receptors were both shown to be resistant to activation by levamisole and sensitive to nicotine. Levamisole antagonised responses to ACh in both ACR-16 and α7 receptors. Both receptors desensitised rapidly and were blocked by dihydro-β- erythroidine. Some pharmacological differences were also shown. The α7 receptor response was blocked by α-bungarotoxin whereas the ACR- 16 receptor was not (Ballivet et al. 1996). Further investigations using the same heterologous expression system showed the anthelmintic 250

Michelle Joyner Conclusion compound oxantel had no agonist effect on ACR-16 recombinant receptors but did antagonise ACh responses. On recombinant chicken α7 receptors, the same compound had low efficiency agonist action. Similar contrasting activity was found with ivermectin which enhanced α7 responses to ACh but attenuated the ACR-16 response to ACh (Raymond et al. 2000).

The reduced response to Bay d9216 was most pronounced in the acr- 16(ok789) strain where no significant effect was found on the locomotion of this strain exposed to 10 – 100 µM Bay d9216 on agar. In these assays some residual, but insignificant activity was found. When exposed to Bay d9216 in liquid no significant effect was seen on the locomotion of this strain except for the first few minutes of exposure. This residual effect on agar and initial effect in liquid is likely to reflect action of Bay d9216 on other receptors which may include ACR-12 and/or ACR-8 containing nAChRs. No effect was seen on the number of progeny produced by this strain when exposed to 200 µM Bay d9216 from the L4 larval stage, this concentration significantly reduced wild type progeny in worms exposed for the same time period. To confirm that ACR-16 containing receptors were the main target for this compound transgenic lines were generated which expressed ACR-16 at the body wall muscle and these lines were shown to have restored susceptibility to Bay d9216.

Receptors containing ACR-16, ACR-12 or ACR-8 subunits have all been shown to be resistant to levamisole (Gottschalk et al. 2005; Touroutine et al. 2005). ACR-16 and ACR-8 have both been shown to be sensitive to nicotine (Gottschalk et al. 2005). This suggests that the nematode nicotine sensitive nAChR might not always be expressed as a homomer consisting of ACR-16 alone, despite the fact that homomeric expression has been successful in Xenopus oocytes.

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Michelle Joyner Conclusion

Bay d9216 has been shown to have activity in two different parasitic worm species of economic importance. Investigations using muscle strips from A. suum muscle showed that Bay d9216 caused contraction which was comparable to the contractile activity of levamisole, as was the desensitisation to increasing concentrations of either compound.

The inhibition of motility in H. contortus caused by both Bay d9216 and levamisole was comparable. These compounds have also been shown to have an inhibitory effect on a strain of H. contortus with multiple anthelmintic resistant. This confirms that these compounds have a different mode of action to ivermectin, members of the benzimidazole family and to the salicylanilide anthelmintic closantel in H. contortus. It would be of interest to expose levamisole resistant H. contortus to Bay d9216 to determine if this compound has resistance breaking potential in this important species of parasitic worm.

In conclusion, the findings presented here clearly show that the acetanilides; amidantel, Bay d9216 and tribendimidine all have anthelmintic potential by disrupting cholinergic signalling, predominantly at the nematode NMJ. Tribendimidine and Bay d9216 have also been shown to inhibit progeny production. Consequently these compounds are likely to impact on more than one essential aspect of the lifecycle of the parasitic worm. Important differences have been revealed between the actions of Bay d9216 and those of levamisole in wild type C. elegans suggesting that these compounds differ in their molecular mode of action. This apparent difference has been further discerned in the reverse genetic screen which has revealed that nAChR subunits which do not contribute to the action of levamisole are implicated in the action of Bay d9216. Therefore Bay d9216 has the potential to break levamisole resistance. The fact that Bay d9216 is likely to have multigenic targets is a positive factor in relation to its anthelmintic potential as the development of resistance to compounds

252

Michelle Joyner Conclusion with a multigenic action tends to be slower than to those with a single molecular target.

Future work

As the data presented here suggest that Bay d9216 targets more than one nAChR subunit. The exposure of a C. elegans strain with mutations in acr-16, acr-12 and acr-8 to Bay d9216 would be informative about the molecular mode of action of this compound. If the triple mutant was completely resistant to the effects of Bay d9216 this would verify the involvement of all three subunits in the action of this compound. It would also be of interest to generate a strain which expresses wild type copies of ACR-16 under a neuronal promoter in an acr-16 mutant background. Exposure of this strain to Bay d9216 would verify if this compound has neuronal activity or is only active at the nematode body wall muscle.

The application of levamisole and Bay d9216 to L-AChRs and N-AChRs heterologously expressed in Xenopus oocytes would serve to elucidate any pharmacological differences between these compounds at the receptor level. It would also be of interest to determine the effects of Bay d9216 on heterologously expressed vertebrate α7 receptors. This would reveal whether or not this compound has selectivity for the nematode receptor and so provide information about selective toxicity.

Bay d9216 and levamisole caused similar contractile responses and desensitisation in A. suum muscle strips. Investigations into the blockade of Bay d9216 and levamisole responses using A. suum muscle strips previously bathed in selective cholinergic antagonists may prove beneficial in discerning the mode of action of these compounds in the parasitic worm. An additional way to determine if Bay d9216 has levamisole resistance breaking potential in an economically important species of parasitic worm would be to expose a levamisole resistant isolate of H. contortus to Bay d9216 253

Michelle Joyner Conclusion

In this project the N-AChR containing ACR-16 has been shown to be the primary target for Bay d9216. Apart from its potential as an anthelmintic compound, Bay d9216 could also provide a novel route to probe the Wnt regulated signalling pathway in C. elegans. Little is known about the regulation and translocation of N-AChRs to the plasma membrane. Recently the Wnt signalling pathway has been found to be involved in this process in C. elegans. The term Wnt is derived from Int (integration) and Wg (wingless) in Drosophila (Klaus and Birchmeier 2008).

Receptor tyrosine kinase-like orphan receptor (ROR) proteins were described as orphan receptors as originally their ligand and signalling pathway were unknown but it has recently been established that ROR proteins are Wnt receptors in multiple species (Green et al. 2008). ROR receptor tyrosine kinases (RTK) are widely expressed in the nervous systems of organisms ranging from humans and mice to Drosophila and C. elegans (Forrester 2002). ROR-RTK proteins can repress transcription of Wnt target genes or modulate Wnt signalling by sequestering Wnt ligands (Green et al. 2008). In C. elegans cam-1 encodes a ROR-RTK which is required for N-AChR signalling (Francis et al. 2005).

Wnt signalling pathways are best characterised for their role in development however, in C. elegans Wnt signalling also regulates localisation of ACR-16 containing nAChRs to the synapse. Mutations in components of the Wnt signalling pathway including cwn-2, (Wnt ligand) or cam-1 (ROR-RTK) result in subsynaptic accumulations of ACR-16 and a reduction in the nicotinic current (Francis et al. 2005; Jensen et al. 2012)

The Wnt signalling pathway at the C. elegans NMJ involves the assembly of CAM-1 & LIN-17 (A frizzled receptor) in the muscle membrane to form a functional heteromer. The Wnt ligand, CWN-2 signals via this heteromeric CAM-1/LIN-17 receptor to activate an intracellular signalling molecule, DSH-1 (dishevelled). Activation of DSH-1 causes the rapid

254

Michelle Joyner Conclusion translocation of ACR-16 receptors to the postsynaptic membrane (Jensen et al. 2012). This pathway is summarised in figure 90.

Figure 90: Wnt signalling pathway regulates N-AChR signalling in C. elegans

CWN-2 - Wnt ligand

CAM-1 – ROR-RTK (Ig – immunoglobulin-like domain, CR – cysteine rich Frizzled domain, Kr – kringle domain, TKD – tyrosine kinase domain)

LIN-17 – Frizzled receptor

DSH-1 – Intracellular signalling molecule (dishevelled)

(Information from Jensen et al. 2012)

255

Michelle Joyner Conclusion

The recently identified Wnt signalling pathway in C. elegans (Jensen et al. 2012) enables the regulation of strength of nicotinic transmission at the NMJ and enables experience dependent plasticity. This pathway is specific to nicotine sensitive N-AChRs. Levamisole sensitive L-AChRs and GABAergic receptors are not affected. Therefore Bay d9216 could be useful in the investigation of the role of ACR-16 in this pathway.

256

Michelle Joyner References References

Agrawal, N., P. V. N. Dasaradhi, A. Mohmmed, P. Malhotra, R. K. Bhatnagar and S. K. Mukherjee (2003). "RNA interference: Biology, mechanism, and applications." Microbiology and Molecular Biology Reviews 67(4): 657.

Albertson, D. G. and J. N. Thomson (1976). "The pharynx of Caenorhabditis elegans." Philosophical transactions of the Royal Society of London. Series B, Biological sciences 275(938): 299-325.

Alkema, M. J., M. Hunter-Ensor, N. Ringstad and H. R. Horvitz (2005). "Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system." Neuron 46(2): 247-260.

Altun, Z. F. and D. H. Hall, Eds. (2011). Nervous system, general description. In WormAtlas.

Altun, Z. F. and D. H. Hall, Eds. (2012). Handbook of C. elegans anatomy. In WormAtlas. Wormatlas.

Arneric, S. P., M. Holladay and M. Williams (2007). "Neuronal nicotinic receptors: A perspective on two decades of drug discovery research." Biochemical Pharmacology 74: 1092-1101.

Avery, L. and H. R. Horvitz (1990). "Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans." The Journal of Experimental Zoology 253(3): 263-270.

Avery, L. and J. H. Thomas (1997). Feeding and Defecation. C. elegans II. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess. Cold Spring Harbor (NY).

Avery, L. and Y. J. You (2012). "C. elegans feeding." WormBook : the online review of C. elegans biology: 1-23.

Ballivet, M., C. Alliod, S. Bertrand and D. Bertrand (1996). "Nicotinic acetylcholine receptors in the nematode Caenorhabditis elegans." J Mol Biol 258(2): 261-269.

Barik, J. and S. Wonnacott (2009). "Molecular and cellular mechanisms of action of nicotine in the CNS." Handbook of Experimental Pharmacology(192): 173-207.

Bartos, M., D. Rayes and C. Bouzat (2006). "Molecular determinants of pyrantel selectivity in nicotinic receptors." Mol Pharmacol 70(4): 1307- 1318. 257

Michelle Joyner References

Ben Arous, J., S. Laffont and D. Chatenay (2009). "Molecular and Sensory Basis of a Food Related Two-State Behavior in C. elegans." Plos One 4(10).

Berezikov, E., C. I. Bargmann and R. H. A. Plasterk (2004). "Homologous gene targeting in Caenorhabditis elegans by biolistic transformation." Nucleic Acids Research 32(4).

Besier, R. B. and S. C. J. Love (2003). "Anthelmintic resistance in sheep nematodes in Australia: the need for new approaches." Australian Journal of Experimental Agriculture 43(12): 1383-1391.

Blane, D., Ed. (1826). The Outlines of the Veterinary Art, 3rd edition, Compton and Ritchie Printers.

Blaxter, M. L., P. De Ley, J. R. Garey, L. X. Liu, P. Scheldeman, A. Vierstraete, J. R. Vanfleteren, et al. (1998). "A molecular evolutionary framework for the phylum Nematoda." Nature 392(6671): 71-75.

Boulin, T., A. Fauvin, C. L. Charvet, J. Cortet, J. Cabaret, J. L. Bessereau and C. Neveu (2011). "Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance." Br J Pharmacol 164(5): 1421-1432.

Boulin, T., M. Gielen, J. E. Richmond, D. C. Williams, P. Paoletti and J. L. Bessereau (2008). "Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor." Proc Natl Acad Sci U S A 105(47): 18590-18595.

Brejc, K., W. J. van Dijk, R. V. Klaassen, M. Schuurmans, J. van der Oost, A. B. Smit and T. K. Sixma (2001). "Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors." Nature 411(6835): 269-276.

Brenner, S. (1973) "The genetics of Caenorhabditis elegans."

Brockie, P. J., D. M. Madsen, Y. Zheng, J. Mellem and A. V. Maricq (2001). "Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42." The Journal of Neuroscience 21(5): 1510-1522.

Brockie, P. J. and A. V. Maricq, Eds. (2006). Ionotropic glutamate receptors: genetics, behavior and electrophysiology. WormBook.

258

Michelle Joyner References

Brown, L. A., A. K. Jones, S. D. Buckingham, C. J. Mee and D. B. Sattelle (2006). "Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study." Int J Parasitol 36(6): 617-624.

Buckingham, S. D. and D. B. Sattelle (2008). "Strategies for automated analysis of C. elegans locomotion." Invertebrate Neuroscience 8(3): 121- 131.

Buckingham, S. D. and D. B. Sattelle (2009). "Fast, automated measurement of nematode swimming (thrashing) without morphometry." BMC neuroscience 10: 84.

Bull, K., A. Cook, N. A. Hopper, A. Harder, L. Holden-Dye and R. J. Walker (2007). "Effects of the novel anthelmintic emodepside on the locomotion, egg-laying behaviour and development of Caenorhabditis elegans." International Journal for Parasitology 37(6): 627-636.

Campos, A., M. P. Cummings, J. L. Reyes and J. P. Laclette (1998). "Phylogenetic relationships of platyhelminthes based on 18S ribosomal gene sequences." Molecular Phylogenetics and Evolution 10(1): 1-10.

Chalfie, M., J. E. Sulston, J. G. White, E. Southgate, J. N. Thomson and S. Brenner (1985). "The Neural Circuit for Touch Sensitivity in Caenorhabditis elegans." Journal of Neuroscience 5(4): 956-964.

Changeux, J. P. (1979). "The acetylcholine receptor: an "allosteric" membrane protein." Harvey lectures 75: 85-254.

Chase, D. L. and M. R. Koelle, Eds. (2007). Biogenic amine neurotransmitters in C. elegans. WormBook, WormBook.

Cinar, H., S. Keles and Y. S. Jin (2005). "Expression profiling of GABAergic motor neurons in Caenorhabditis elegans." Current Biology 15(4): 340-346.

Cohen, M., V. Reale, B. Olofsson, A. Knights, P. Evans and M. de Bono (2009). "Coordinated Regulation of Foraging and Metabolism in C- elegans by RFamide Neuropeptide Signaling." Cell Metabolism 9(4): 375- 385.

Cooper, O. (2012). Anthelmintic resistance continues to hamper UK farmers. Farmer's Weekly. UK.

Corringer, P. J., N. Le Novere and J. P. Changeux (2000). "Nicotinic receptors at the amino acid level." Annual Review of Pharmacology and Toxicology 40: 431-458.

259

Michelle Joyner References

Croll, N. A. M. B. E., Ed. (1977). Biology of Nematodes.

Culetto, E., H. A. Baylis, J. E. Richmond, A. K. Jones, J. T. Fleming, M. D. Squire, J. A. Lewis, et al. (2004). "The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit." The Journal of biological chemistry 279(41): 42476- 42483.

Cully, D. F., P. S. Paress, K. K. Liu, J. M. Schaeffer and J. P. Arena (1996). "Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin." Journal of Biological Chemistry 271(33): 20187-20191.

Cully, D. F., D. K. Vassilatis, K. K. Liu, P. S. Paress, L. H. T. Vanderploeg, J. M. Schaeffer and J. P. Arena (1994). "Cloning of an Avermectin- Sensitive Glutamate-Gated Chloride Channel from Caenorhabditis elegans." Nature 371(6499): 707-711. de Bono, M. and A. V. Maricq (2005). "Neuronal substrates of complex behaviors in C. elegans." Annual Review of Neuroscience 28: 451-501.

Dolgin, E. S., M. A. Felix and A. D. Cutter (2008). "Hakuna Nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C briggsae." Heredity 100(3): 304-315.

Ducray, P., N. Gauvry, F. Pautrat, T. Goebel, J. Fruechtel, Y. Desaules, S. S. Weber, et al. (2008). "Discovery of amino-acetonitrile derivatives, a new class of synthetic anthelmintic compounds." Bioorganic & Medicinal Chemistry Letters 18(9): 2935-2938.

Dykxhoorn, D. M. and J. Lieberman (2005). "The silent revolution: RNA interference as basic biology, research tool, and therapeutic." Annual Review of Medicine 56: 401-423.

Eimer, S., A. Gottschalk, M. Hengartner, H. R. Horvitz, J. Richmond, W. R. Schafer and J. L. Bessereau (2007). "Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50." Embo Journal 26: 4313-4323. 260

Michelle Joyner References

Faust, E. C. (1939). Human Helminthology. London, Henry Kimpton.

Felix, M.-A. (2008). "RNA interference in nematodes and the chance that favored Sydney Brenner." Journal of Biology 7: 1197.

Fleming, J. T., M. D. Squire, T. M. Barnes, C. Tornoe, K. Matsuda, J. Ahnn, A. Fire, et al. (1997). "Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits." J Neurosci 17(15): 5843-5857.

Flibotte, S., M. L. Edgley, I. Chaudhry, J. Taylor, S. E. Neil, A. Rogula, R. Zapf, et al. (2010). "Whole-Genome Profiling of Mutagenesis in Caenorhabditis elegans." Genetics 185(2): 431-441.

Forrester, W. C. (2002). "The Ror receptor tyrosine kinase family." Cellular and Molecular Life Sciences 59(1): 83-96.

Fox, R. M., S. E. Von Stetina, S. J. Barlow, C. Shaffer, K. L. Olszewski, J. H. Moore, D. Dupuy, et al. (2005). "A gene expression fingerprint of C. elegans embryonic motor neurons." BMC Genomics 6.

Francis, M. M., S. P. Evans, M. Jensen, D. M. Madsen, J. Mancuso, K. R. Norman and A. V. Maricq (2005). "The Ror receptor tyrosine kinase CAM- 1 is required for ACR-16-mediated synaptic transmission at the C. elegans neuromuscular junction." Neuron 46(4): 581-594.

Gendrel, M., G. Rapti, J. E. Richmond and J. L. Bessereau (2009). "A secreted complement-control-related protein ensures acetylcholine receptor clustering." Nature 461(7266): 992-996.

Ghedin, E. and B. P. Anton (2009). "Draft genome of the filarial nematode parasite Brugia malayi, (vol 317, pg 1756, 2007)." Science 323(5910): 38-38.

Girard, L. R., T. J. Fiedler, T. W. Harris, F. Carvalho, I. Antoshechkin, M. Han, P. W. Sternberg, et al. (2007). "WormBook: the online review of Caenorhabditis elegans biology." Nucleic Acids Research 35: D472- D475.

Gossner, A. G., V. M. Venturina, D. J. Shaw, J. M. Pemberton and J. Hopkins (2012). "Relationship between susceptibility of Blackface sheep to Teladorsagia circumcincta infection and an inflammatory mucosal T cell response." Veterinary Research 43(1): 26.

Gottschalk, A., R. B. Almedom, T. Schedletzky, S. D. Anderson, J. R. Yates and W. R. Schafer (2005). "Identification and characterization of

261

Michelle Joyner References novel nicotinic receptor-associated proteins in Caenorhabditis elegans." Embo J 24(14): 2566-2578.

Grando, S. A., D. A. Kist, M. Qi and M. V. Dahl (1993). "Human keratinocytes synthesize, secrete, and degrade acetylcholine." The Journal of Investigative Dermatology 101(1): 32-36.

Green, J. L., S. G. Kuntz and P. W. Sternberg (2008). "Ror receptor tyrosine kinases: orphans no more." Trends in Cell Biology 18(11): 536- 544.

Gryseels, B., K. Polman, J. Clerinx and L. Kestens (2006). "Human schistosomiasis." Lancet 368(9541): 1106-1118.

Guest, M., K. Bull, R. J. Walker, K. Amliwala, V. O'Connor, A. Harder, L. Holden-Dye, et al. (2007). "The calcium-activated potassium channel, SLO-1, is required for the action of the novel cyclo-octadepsipeptide anthelmintic, emodepside, in Caenorhabditis elegans." International Journal for Parasitology 37: 1577-1588.

Guo, R. C., G. Y. Yuan, B. J. Wang, C. M. Wei and R. Zhang (2009). "Disposition and metabolism of tribendimidine in Chinese human." Journal of Pharmacological Sciences 109: 244p-244p.

Halevi, S., J. McKay, M. Palfreyman, L. Yassin, M. Eshel, E. Jorgensen and M. Treinin (2002). "The C-elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors." Embo Journal 21(5): 1012-1020.

Halevi, S., J. McKay, M. Palfreyman, L. Yassin, M. Eshel, E. Jorgensen and M. Treinin (2002). "The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors." Embo J 21(5): 1012-1020.

Hall, L. R. and E. Pearlman (1999). "Pathogenesis of onchocercal keratitis (river blindness)." Clinical Microbiology Reviews 12(3): 445-+.

Hall, S. from http://www.abdn.ac.uk/organic/index.php.

Hallam, S., E. Singer, D. Waring and Y. Jin (2000). "The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification." Development 127(19): 4239-4252.

Harder, A. (2002). "Milestones of helmintic research at Bayer." Parasitol Res 88(5): 477-480.

Harder, A., H. P. Schmitt-Wrede, J. Krucken, P. Marinovski, F. Wunderlich, J. Willson, K. Amliwala, et al. (2003). "Cyclooctadepsipeptides - an anthelmintically active class of compounds exhibiting a novel mode of action." International Journal of Antimicrobial Agents 22(3): 318-331. 262

Michelle Joyner References

Haspel, G., M. J. O'Donovan and A. C. Hart (2010). "Motoneurons dedicated to either forward or backward locomotion in the nematode Caenorhabditis elegans." The Journal of Neuroscience 30(33): 11151- 11156.

Hassoni, A. A., G. A. Kerkut and R. J. Walker (1988). "Evidence That Levamisole, Pyrantel, Morantel, Amidantel, Deacylated Amidantel and Hycanthone May Act on Acetycholine Receptors of Central Neurons of Helix-Aspersa." Comparative Biochemistry and Physiology C- Pharmacology Toxicology & Endocrinology 91(2): 525-533.

Hassoni, A. A., G. A. Kerkut and R. J. Walker (1988). "Evidence That Levamisole, Pyrantel, Morantel, Amidantel, Deacylated Amidantel and Hycanthone May Act on Acetycholine Receptors of Central Neurons of Helix aspersa." Comparative Biochemistry and Physiology C- Pharmacology Toxicology & Endocrinology 91(2): 525-533.

Haugstetter, J., T. Blicher and L. Ellgaard (2005). "Identification and characterization of a novel thioredoxin-related transmembrane protein of the endoplasmic reticulum." The Journal of Biological Chemistry 280(9): 8371-8380.

Hills, T., P. J. Brockie and A. V. Maricq (2004). "Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans." The Journal of Neuroscience 24(5): 1217-1225.

Holden-Dye, L. and R. J. Walker (2007). "Anthelmintic drugs." WormBook: 1-13.

Horvitz, H. R., M. Chalfie, C. Trent, J. E. Sulston and P. D. Evans (1982). "Serotonin and octopamine in the nematode Caenorhabditis elegans." Science 216(4549): 1012-1014.

Hotez, P. J., P. J. Brindley, J. M. Bethony, C. H. King, E. J. Pearce and J. Jacobson (2008). "Helminth infections: the great neglected tropical diseases." Journal of Clinical Investigation 118(4): 1311-1321.

Hu, Y., S. H. Xiao and R. V. Aroian (2009). "The new anthelmintic tribendimidine is an L-type (levamisole and pyrantel) nicotinic acetylcholine receptor agonist." Plos Negl Trop Dis 3(8).

Jagannathan, S., D. L. Laughton, C. L. Critten, T. M. Skinner, L. Horoszok and A. J. Wolstenholme (1999). "Ligand-gated chloride channel subunits encoded by the Haemonchus contortus and Ascaris suum orthologues of the Caenorhabditis elegans gbr-2 (avr-14) gene." Molecular and Biochemical Parasitology 103(2): 129-140.

263

Michelle Joyner References

Jensen, M., F. J. Hoerndli, P. J. Brockie, R. Wang, E. Johnson, D. Maxfield, M. M. Francis, et al. (2012). "Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system." Cell 149(1): 173-187.

Jones, A. K., P. Davis, J. Hodgkin and D. B. Sattelle (2007). "The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans: an update on nomenclature." Invert Neurosci 7(2): 129-131.

Jones, A. K. and D. B. Sattelle (2004). "Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans." Bioessays 26(1): 39-49.

Jones, J., R. Pearson and S. Jeckel (2012). "Suspected anthelmintic resistance to macrocyclic lactones in lambs in the UK." The Veterinary Record 170(2): 59-60.

Kagawa, H., K. Takuwa and Y. Sakube (1997). "Mutations and expressions of the tropomyosin gene and the troponin C gene of Caenorhabditis elegans." Cell Structure and Function 22(1): 213-218.

Kaminsky, R., P. Ducray, M. Jung, R. Clover, L. Rufener, J. Bouvier, S. S. Weber, et al. (2008). "A new class of anthelmintics effective against drug-resistant nematodes." Nature 452(7184): 176-U119.

Kaminsky, R., N. Gauvry, S. S. Weber, T. Skripsky, J. Bouvier, A. Wenger, F. Schroeder, et al. (2008). "Identification of the amino-acetonitrile derivative monepantel (AAD 1566) as a new anthelmintic drug development candidate." Parasitology Research 103(4): 931-939.

Keating, B. A., P. S. Carberry, P. S. Bindraban, S. Asseng, H. Meinke and J. Dixon (2010). "Eco-efficient Agriculture: Concepts, Challenges, and Opportunities." Crop Science 50(2): S109-S119.

Keiser, J., X. Shu-Hua and J. Utzinger (2006). "Effect of tribendimidine on adult Echinostoma caproni harbored in mice, including scanning electron microscopic observations." Journal of Parasitology 92(4): 858- 862.

Keiser, J., K. Thiemann, Y. Endriss and J. Utzinger (2008). "Strongyloides ratti: In Vitro and In Vivo Activity of Tribendimidine." Plos Neglected Tropical Diseases 2(1).

Keiser, J. and J. Utzinger (2008). "Efficacy of current drugs against soil- transmitted helminth infections - Systematic review and meta-analysis." Journal of the American Medical Association 299(16): 1937-1948.

264

Michelle Joyner References

Keiser, J. and J. Utzinger (2010). "The Drugs We Have and the Drugs We Need Against Major Helminth Infections." Advances in Parasitology, Vol 73 73: 197-230.

Keiser, J., J. Utzinger, S. H. Xiao, P. Odermatt and S. Tesana (2008). "Opisthorchis viverrini: efficacy and tegumental alterations following administration of tribendimidine in vivo and in vitro." Parasitology Research 102(4): 771-776.

Kim, J., D. S. Poole, L. E. Waggoner, A. Kempf, D. S. Ramirez, P. A. Treschow and W. R. Schafer (2001). "Genes affecting the activity of nicotinic receptors involved in Caenorhabditis elegans egg-laying behavior." Genetics 157(4): 1599-1610.

Klaus, A. and W. Birchmeier (2008). "Wnt signalling and its impact on development and cancer." Nature Reviews 8(5): 387-398.

Le Jambre, L. F., J. H. Gill, I. J. Lenane and E. Lacey (1995). "Characterisation of an avermectin resistant strain of Australian Haemonchus contortus." International Journal for Parasitology 25(6): 691-698.

Lena, C. and J. P. Changeux (1997). "Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders." Current Opinion in Neurobiology 7(5): 674-682.

Lewis, J. A., C. H. Wu, H. Berg and J. H. Levine (1980). "The genetics of levamisole resistance in the nematode Caenorhabditis elegans." Genetics 95(4): 905-928.

Lewis, J. A., C. H. Wu, J. H. Levine and H. Berg (1980). "Levamisole- Resistant Mutants of the Nematode Caenorhabditis elegans Appear to Lack Pharmacological Acetylcholine-Receptors." Neurosci 5(6): 967-989.

Li, C. and K. Kim (2008). "Neuropeptides." WormBook: 1-36.

Li, C., K. Kim and L. S. Nelson (1999). "FMRFamide-related neuropeptide gene family in Caenorhabditis elegans." Brain Research 848(1-2): 26-34.

Lockery, S. R., S. E. Hulme, W. M. Roberts, K. J. Robinson, A. Laromaine, T. H. Lindsay, G. M. Whitesides, et al. (2012). "A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans." Lab on a chip 12(12): 2211-2220.

Lustigman, S., R. K. Prichard, A. Gazzinelli, W. N. Grant, B. A. Boatin, J. S. McCarthy and M. G. Basanez (2012). "A research agenda for helminth diseases of humans: the problem of helminthiases." Plos Neglected Tropical Diseases 6(4): e1582. 265

Michelle Joyner References

Malikides, N., K. Spencer, R. Mahoney, K. Baker, K. Vanhoff, C. Hall, R. Debenedetti, et al. (2009). "Safety of an amino-acetonitrile derivative (AAD), monepantel, in ewes and their offspring following repeated oral administration." New Zealand Veterinary Journal 57(4): 193-202.

Martin, R. J. (1985). "gamma-Aminobutyric acid- and piperazine-activated single-channel currents from Ascaris suum body muscle." British Journal of Pharmacology 84(2): 445-461.

Martin, R. J. (1997). "Modes of action of anthelmintic drugs." Veterinary Journal 154(1): 11-34.

Martinez-Valladares, M., J. M. Martinez-Perez, D. Robles-Perez, C. Cordero-Perez, M. R. Famularo, N. Fernandez-Pato, L. Castanon-Ordonez, et al. (2012). "The present status of anthelmintic resistance in gastrointestinal nematode infections of sheep in the northwest of Spain by in vivo and in vitro techniques." Veterinary Parasitology.

Maryon, E. B., R. Coronado and P. Anderson (1996). "unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction." The Journal of Cell Biology 134(4): 885-893.

Matta, S. G., D. J. Balfour, N. L. Benowitz, R. T. Boyd, J. J. Buccafusco, A. R. Caggiula, C. R. Craig, et al. (2007). "Guidelines on nicotine dose selection for in vivo research." Psychopharmacology 190(3): 269-319.

McIntire, S. L., E. Jorgensen and H. R. Horvitz (1993). "Genes required for GABA function in Caenorhabditis elegans." Nature 364(6435): 334-337.

McKay, J. P., D. M. Raizen, A. Gottschalk, W. R. Schafer and L. Avery (2004). "eat-2 and eat-18 are required for nicotinic neurotransmission in the Caenorhabditis elegans pharynx." Genetics 166(1): 161-169.

McKellar, Q. A. and F. Jackson (2004). "Veterinary anthelmintics: old and new." Trends Parasitol 20(10): 456-461.

Mello, C. C., J. M. Kramer, D. Stinchcomb and V. Ambros (1991). "Efficient Gene-Transfer in C-Elegans - Extrachromosomal Maintenance and Integration of Transforming Sequences." Embo Journal 10(12): 3959-3970.

Miller, C. M., T. S. Waghorn, D. M. Leathwick, P. M. Candy, A. M. Oliver and T. G. Watson (2012). "The production cost of anthelmintic resistance in lambs." Veterinary Parasitology 186(3-4): 376-381.

266

Michelle Joyner References

Mongan, N. P., A. K. Jones, G. R. Smith, M. S. Sansom and D. B. Sattelle (2002). "Novel alpha7-like nicotinic acetylcholine receptor subunits in the nematode Caenorhabditis elegans." Protein Sci 11(5): 1162-1171.

Muller, R. (1975). Worms and disease. London, William Heinmann Medical Books Ltd.

Nathoo, A. N., R. A. Moeller, B. A. Westlund and A. C. Hart (2001). "Identification of neuropeptide-like protein gene families in Caenorhabditiselegans and other species." Proceedings of the National Academy of Sciences of the United States of America 98(24): 14000- 14005.

Nayak, S., J. Goree and T. Schedl (2005). "fog-2 and the evolution of self- fertile hermaphroditism in Caenorhabditis." PLoS Biology 3(1): e6.

Neveu, C., C. L. Charvet, A. Fauvin, J. Cortet, R. N. Beech and J. Cabaret (2010). "Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance." Pharmacogenet Genomics 20(7): 414-425.

Njue, A. I. and R. K. Prichard (2004). "Efficacy of ivermectin in calves against a resistant Cooperia oncophora field isolate." Parasitology Research 93(5): 419-422.

O'Connor, L. J., S. W. Walkden-Brown and L. P. Kahn (2006). "Ecology of the free-living stages of major trichostrongylid parasites of sheep." Veterinary Parasitology 142(1-2): 1-15.

Olsen, O. W. (1986). Animal parasites.

Papke, R. L., L. P. Dwoskin and P. A. Crooks (2007). "The pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery." Journal of Neurochemistry 101(1): 160-167.

Pennington, A. J. and R. J. Martin (1990). "A patch-clamp study of acetylcholine-activated ion channels in Ascaris suum muscle." The Journal of Experimental Biology 154: 201-221.

Perry, B. D., R. F. Randolph, J. J. McDermott, K. R. Sones and P. K. Thornton (2002). Investing in animal health research to alleviate poverty. I. L. R. Institute. Nairobi, Kenya.

Peter, J. W. and P. Chandrawathani (2005). "Haemonchus contortus: parasite problem No. 1 from tropics - Polar Circle. Problems and prospects for control based on epidemiology." Tropical Biomedicine 22(2): 131-137. 267

Michelle Joyner References

Pierce, S. B., M. Costa, R. Wisotzkey, S. Devadhar, S. A. Homburger, A. R. Buchman, K. C. Ferguson, et al. (2001). "Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family." Genes & Development 15(6): 672-686.

Prichard, R. (1994). "Anthelmintic Resistance." Veterinary Parasitology 54(1-3): 259-268.

Prichard, R. K. and T. G. Geary (2008). "Drug discovery: Fresh hope to can the worms." Nature 452(7184): 157-158.

Qian, H., R. J. Martin and A. P. Robertson (2006). "Pharmacology of N-, L- , and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum." Faseb Journal 20(14): 2606-+.

Qian, H., R. J. Martin and A. P. Robertson (2006). "Pharmacology of N-, L- , and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum." Faseb J 20: 2606.

Qian, H., A. P. Robertson, J. A. Powell-Coffman and R. J. Martin (2008). "Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans." Faseb J 22(9): 3247-3254.

Raizen, D. M., R. Y. Lee and L. Avery (1995). "Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans." Genetics 141(4): 1365-1382.

Rand, J. B., Ed. (2007). Acetylcholine. WormBook.

Raymond, V., N. P. Mongan and D. B. Sattelle (2000). "Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken alpha7 and ACR-16 from the nematode Caenorhabditis elegans." Neurosci 101(3): 785-791.

Richmond, J. E. and E. M. Jorgensen (1999). "One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction." Nature Neurosci 2(9): 791-797.

Riddle DL, B. T., Meyer BJ & Priess JR, Ed. (1977). C. elegans II.

Rim, H., K. Joo, Y. Kim, J. Lee and S. Song (1980). "Anthelmintic effect of Amidantel (Bay d8815) against Ancylostoma duodenale infectio." Korean Journal of Parasitology 18(1): 24-36.

Riou, M., F. Guegnard, Y. Le Vern and D. Kerboeuf (2003). "Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus 268

Michelle Joyner References contortus by changing cholesterol content: effects on resistance to anthelmintics." The Journal of Antimicrobial Chemotherapy 52(2): 180- 187.

Robertson, A. P., C. L. Clark, T. A. Burns, D. P. Thompson, T. G. Geary, S. M. Trailovic and R. J. Martin (2002). "Paraherquamide and 2-deoxy- paraherquamide distinguish cholinergic receptor subtypes in Ascaris muscle." J Pharmacol Exp Ther 302(3): 853-860.

Robertson, A. P. and R. J. Martin (2007). "Ion-channels on parasite muscle: pharmacology and physiology." Invert Neurosci 7(4): 209-217.

Robertson, S. J. and R. J. Martin (1993). "Levamisole-activated single- channel currents from muscle of the nematode parasite Ascaris suum." British Journal of Pharmacology 108(1): 170-178.

Rougvie, A. E. (2005). "Intrinsic and extrinsic regulators of developmental timing: from miRNAs to nutritional cues." Development 132(17): 3787-3798.

Ruaud, A. F. and J. L. Bessereau (2006). "Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C-elegans." Development 133(11): 2211-2222.

Ruaud, A. F. and J. L. Bessereau (2006). "Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans." Development 133(11): 2211-2222.

Rufener, L., J. Keiser, R. Kaminsky, P. Maser and D. Nilsson (2010). "Phylogenomics of ligand-gated ion channels predicts monepantel effect." PLoS Pathog 6(9).

Rufener, L., P. Maser, I. Roditi and R. Kaminsky (2009). "Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel." Plos Pathog 5(4): e1000380.

Rufener, L., P. Maser, I. Roditi and R. Kaminsky (2009). "Haemonchus contortus Acetylcholine Receptors of the DEG-3 Subfamily and Their Role in Sensitivity to Monepantel." Plos Pathogens 5(4): -.

Sano, T., Y. Tabuse, K. Nishiwaki and J. Miwa (1995). "The tpa-1 gene of Caenorhabditis elegans encodes two proteins similar to Ca(2+)- independent protein kinase Cs: evidence by complete genomic and complementary DNA sequences of the tpa-1 gene." Journal of Molecular Biology 251(4): 477-485.

Sanyal, S., R. F. Wintle, K. S. Kindt, W. M. Nuttley, R. Arvan, P. Fitzmaurice, E. Bigras, et al. (2004). "Dopamine modulates the plasticity 269

Michelle Joyner References of mechanosensory responses in Caenorhabditis elegans." The EMBO Journal 23(2): 473-482.

Sattelle, D. B. (2009). "Invertebrate nicotinic acetylcholine receptors- targets for chemicals and drugs important in agriculture, veterinary medicine and human health." Journal of Pesticide Science 34(4): 233- 240.

Schafer, W. R., Ed. (2005). Wormbook. The C. elegans Research Community

Schuske, K., A. A. Beg and E. M. Jorgensen (2004). "The GABA nervous system in C. elegans." Trends in Neurosciences 27(7): 407-414.

Schuske, K., M. T. Palfreyman, S. Watanabe and E. M. Jorgensen (2007). "UNC-46 is required for trafficking of the vesicular GABA transporter." Nature Neuroscience 10(7): 846-853.

Scott, A. L. and E. Ghedin (2009). "The genome of Brugia malayi - all worms are not created equal." Parasitol Int 58(1): 6-11.

Scott, A. L. and E. Ghedin (2009). "The genome of Brugia malayi - All worms are not created equal." Parasitology International 58(1): 6-11.

Shoop, W. L., H. W. Haines, C. H. Eary and B. F. Michael (1992). "Acute Toxicity of Paraherquamide and Its Potential as an Anthelmintic." American Journal of Veterinary Research 53(11): 2032-2034.

Singson, A. (2001). "Every sperm is sacred: fertilization in Caenorhabditis elegans." Developmental Biology 230(2): 101-109.

Skelly, P. (2008). "Fighting killer worms." Scientific American 298(5): 94- 99.

Song, B. M. and L. Avery (2012). "Serotonin activates overall feeding by activating two separate neural pathways in Caenorhabditis elegans." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(6): 1920-1931.

Stein, L. D., Z. Bao, D. Blasiar, T. Blumenthal, M. R. Brent, N. Chen, A. Chinwalla, et al. (2003). "The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics." PLoS Biology 1(2): E45.

Stroud, R. M. and J. Finermoore (1985). "Acetylcholine receptor structure, function and evolution." Annual Review of Cell Biology 1: 317- 351.

270

Michelle Joyner References

Stubbings, L. (2011). Worms in sheep. Farmer's weekly.

Suarez, V. H. and S. L. Cristel (2007). "Anthelmintic resistance in cattle nematode in the western Pampeana Region of Argentina." Veterinary Parasitology 144(1-2): 111-117.

Sulston, J. E. and S. Brenner (1973) "The DNA of Caenorhabditis elegans."

Thierry-Mieg, D. and J. Thierry-Mieg (2006). "AceView: a comprehensive cDNA-supported gene and transcripts annotation." Genome Biology 7 Suppl 1: S12 11-14.

Thomas, H. (1979). "Efficacy of Amidantel, a New Anthelmintic, on Hookworms and Ascarids in Dogs." Tropenmed Parasitol 30(3): 404-408.

Tobin, D. V. and R. M. Saito (2012). "Developmental decisions: balancing genetics and the environment by C. elegans." Cell Cycle 11(9): 1666- 1671.

Tomlinson, G., C. A. Albuquerque and R. A. Woods (1985). "The Effects of Amidantel (Bay d8815) and Its Deacylated Derivative (Bay d9216) on Caenorhabditis-Elegans." European Journal of Pharmacology 113(2): 255- 262.

Touroutine, D., R. M. Fox, S. E. Von Stetina, A. Burdina, D. M. Miller and J. E. Richmond (2005). "acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction." J Biol Chem 280(29): 27013-27021.

Towers, P. R., B. Edwards, J. E. Richmond and D. B. Sattelle (2005). "The Caenorhabditis elegans lev-8 gene encodes a novel type of nicotinic acetylcholine receptor alpha subunit." J Neurochem 93(1): 1-9.

Trent, C., N. Tsuing and H. R. Horvitz (1983). "Egg-laying defective mutants of the nematode Caenorhabditis elegans." Genetics 104(4): 619-647.

Von Stetina, S. E., J. D. Watson, R. M. Fox, K. L. Olszewski, W. C. Spencer, P. J. Roy and D. M. Miller (2007). "Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C-elegans nervous system." Genome Biology 8(7).

Wang, J., B. Czech, A. Crunk, A. Wallace, M. Mitreva, G. J. Hannon and R. E. Davis (2011). "Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles." Gen Res 21(9): 1462-1477.

271

Michelle Joyner References

Ward, S. and J. S. Carrel (1979). "Fertilization and Sperm Competition in the Nematode Caenorhabditis elegans." Developmental Biology 73(2): 304-321.

Wessler, I. and C. J. Kirkpatrick (2008). "Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans." British Journal of Pharmacology 154(8): 1558-1571.

White, J. G., E. Southgate, J. N. Thomson and S. Brenner (1986). "The structure of the nervous system of the nematode Caenorhabditis elegans." Philosophical transactions of the Royal Society of London. Series B, Biological sciences 314(1165): 1-340.

WHO. (2004). "Global Burden of Disease." from http://www.who.int/healthinfo/global_burden_disease/2004_report_upd ate/en/.

WHO. (2008). "WHO Weekly epidemiological report." from http://www.who.int/wer/en/.

WHO. (2012). "Weekly Epidemiological Record." vol. 87, 23 Retrieved 05/09/2012, 2012, from http://www.who.int/wer/2012/wer8723/en/index.html.

Williamson, S. M., A. P. Robertson, L. Brown, T. Williams, D. J. Woods, R. J. Martin, D. B. Sattelle, et al. (2009). "The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits." Plos Pathogens 5(7): e1000517.

Williamson, S. M., T. K. Walsh and A. J. Wolstenholme (2007). "The cys- loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans." Invert Neurosci 7(4): 219-226.

Winter, M. D. (2002). "Nematodirus battus 50 years on - a realistic vaccine candidate?" Trends in Parasitology 18(7): PII S1471- 4922(1402)02303-02306.

Wollweber, H., E. Niemers, W. Flucke, P. Andrews, H. P. Schulz and H. Thomas (1979). "Amidantel, a Potent Anthelmintic from a New Chemical Class." Drug Research 29-1(1): 31-32.

Wolstenholme, A. J., I. Fairweather, R. Prichard, G. von Samson- Himmelstjerna and N. C. Sangster (2004). "Drug resistance in veterinary helminths." Trends in Parasitology 20(10): 469-476.

272

Michelle Joyner References

Woods, R. A., K. M. B. Malone, C. A. Albuquerque and G. Tomlinson (1986). "The Effects of Amidantel (Bay d8815) and Its Deacylated Derivative (Bay d9216) on Wild-Type and Resistant Mutants of Caenorhabditis elegans." Canadian Journal of Zoology 64(6): 1310-1316.

Xiao, S. H., H. M. Wu, M. Tanner, J. Utzinger and W. Chong (2005). "Tribendimidine: a promising, safe and broad-spectrum anthelmintic agent from China." Acta Tropica 94(1): 1-14.

Xiao, S. H., J. Xue, L. L. Xu, Q. Zheng, H. Q. Qiang and Y. N. Zhang (2009). "The in vitro and in vivo effect of tribendimidine and its metabolites against Clonorchis sinensis." Parasitology Research 105(6): 1497-1507.

Yuan, G. Y., B. J. Wang, C. M. Wei, R. Zhang and R. C. Guo (2008). "LC-MS determination of p-(1-dimethylamino ethylimino)aniline: A metabolite of tribendimidine in human plasma." Chromatographia 68(1-2): 139-142.

Zia, S., A. Ndoye, V. T. Nguyen and S. A. Grando (1997). "Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells." Research Communications in Molecular Pathology and Pharmacology 97(3): 243- 262.

Zinser, E. W., M. L. Wolf, S. J. Alexander-Bowman, E. M. Thomas, J. P. Davis, V. E. Groppi, B. H. Lee, et al. (2002). "Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals." Journal of Veterinary Pharmacology and Therapeutics 25(4): 241-250.

273