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# General discussion {#discussion}

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## Environmental levels of neonicotinoids do not impact on the behavior or development of *C. elegans*
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Increased use of insecticides requires a better understanding of their environmental impact. Therefore, the initial aim of this project was to investigate the effects of neonicotinoid-insecticides on the Nematoda representative *C. elegans*. Neonicotinoids have been introduced to the marked in the 1990s and since have become the most commonly used insecticides worldwide [@jeschke2011]. They have many advantages, including a high potency against wide range pest insects (Section \@ref(potentpests)) and low mammalian toxicity. However, neonicotinoids can have significant field effects on non-target species. The adverse effects of environmental neonicotinoids on bees have been studied for decades (Section \@ref(sublethalbees)), whereas the effects on other ecologically important species is less understood but unlikely to have a problem.
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There are several forms in which neonicotinoids are delivered onto fields including spray and granules, but seed-coating is the most common method [@jeschke2011]. This focused application ensures the presence of drug inside the plant at effective concentrations [@stamm2016], nevertheless neonicotinoids have long half-life and a leeching potential, therefore can reside in soil for prolonged time periods, coming in contact with inhabiting organisms. This leads to potential exposure to soil namatodes and earth worms, two important cultivars of the biomass and nutrient cyclers, that contribute to the soil fertility [@ingham1985; @neher2001]. The lethal dose of neonicotinoids on earths worms and nematodes varies between 2.74 and 62.08 $\mu$M (Table \@ref(tab:toxallanimal)), however neonicotinoids at lower doses can induce sublethal effects (Section \@ref(sublethalsoilworm)). The investigations into their effects on the nematode representative and model organism *C. elegans* were carried using thiacloprid, clothianidin and nitenpyram. 

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The effects of these three compounds on locomotion in liquid and solid media, egg-laying and egg-hatching of wild-type worm were investigated (Chapter 3 and 4). This study reports low potency of neonicotinoids against wild-type *C. elegans* [@kudelska2017]. Neonicotinoids were either not effective, or effective at mM concentrations. 
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These data suggests that neonicotinoids have minimal, if any, effect on locomotion, reproduction or feeding of *C. elegans*, however it is possible that they affect more intricate aspects of worm’s physiology. They impact on learning and plasticity in honeybees [@williamson2013], therefore efforts into determining their effects on the ability to perceive and process information could be made. For example, decision making could be tested in food leaving [@shtonda2006] or chemotaxis assays [@law2004]. 

<!-- When tested against locomotion in liquid, nitenpyram had an inhibitory effect at mM concentrations and the estimated EC50 of 195.8 mM. Thiacloprid and clothianidin had no effect at concentrations up to 1 and 2.5 nM, respectively (Section \@ref(effectsofneonicsonthrashing)). When on solid medium, the locomotion was inhibited by thiacloprid with the estimated EC50 of 3.7 mM but not by nitenpyram at 1 mM or clothianidin at 3.75 mM (Section \@ref(bodybendsneonics)). The low potency of neonicotinoids on intact *C. elegans* is consistent with the literature (Section \@ref(chapter3effectsofneonics)). -->
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<!-- It would also be beneficial to perform long-term exposure assay to determine whether neonicotinoids bio-accumulate in worm’s adipose tissue, or are metabolised to toxic chemical species [@suchail2001]. However, experiemnts in which Lastly, these compounds can act on multiple targets and exert effects at a very low and high doses [@pisa2015], therefore it would be beneficial to determine if *C. elegans* exposure to low doses (high nM - low $\mu$M) have an impact on its behavior. -->

Exposure of *C. elegans* mutant with increased cuticle permeability [@xiong2017] resulted in increased sensitivity of *C. elegans* to tested compounds. Ten-fold increase in the potency of nitenpyram was noted, as indicated by the shift in the EC50 in the thrashing assay. Thiacloprid and clothianidin were with no effect on the wild-type strain, but inhibited locomotion with the EC50 of 337.6 $\mu$M and and 3.5 mm, respectively (Section \@ref(effectsofneonicsonthrashing)). The nematode cuticle is the major route of entry for many drugs, therefore the ability of compounds to cross the cuticular barrier often defines their potency [@alvarez2007]. The cuticle limits bioavailability of many compound used in agriculture [@xiong2017] and by doing so it protects the worm against their potentially harmful effects. 

The cuticle encapsulates the body of nematodes as well as earthworms. Their structures have several similarities, as revealed by the electron microscopy of redworm *L. terrestris*, greenworm *Allolobophora chlorotica* [@reed1948; @coggeshall1966; @knapp1971] and *C. elegans* preparations [@cox1981]. The main components of the cuticle are the collagen fibrils embedded within the matrix. This relatively thick layer is covered by a much thinner epicuticle, which consists mainly of lipids. In earthworms, the epicuticle is perforated by a coat of outward projections, shorter ellipsoidal bodies and longer microvilli. In *C. elegans* the surface coat consists of the glycoprotein-mesh. Based on the architectural and chemical similarities, it is likely that the cuticle of earthworms may also play a role in drug permeability. This is however poorly investigated. 
<!-- [@carter2014].   -->

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Generally, the concentrations at which neonicotinoids impair on *C. elegans* behaviour are several folds higher than those found in the field [@sanchez-bayo2016] and concentrations effective against insect [@goulson2013]. These data suggests *C. elegans* is not impacted by neonicotinoids in the field, however no field-studies are available to confirm this.
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The sensitivity of *C. elegans* to neonicotinoids differs from that of parasitic nematodes and earth worms. In *C. elegans* neonicotinoids impact on locomotion at low mM concentrations (Figure \@ref(fig:thrashing-tc-comp-label) and \@ref(fig:BB-plot-label)), but have no effects on reproduction (Figure \@ref(fig:EL-plot-label) and \@ref(fig:EH-plot-label)). In plant parasitic nematode, neonicotinoids are effective at $\mu$M concentrations. The LD50 of thiacloprid on the root-knot nematode *M. incognita* is 143.23 $\mu$M [@dong2014], whereas the IC50 from the egg-hatching experiments is 300 $\mu$M [@dong2014; @dong2017]. Earthworms seem to be the most susceptible, with reported toxic lethal doses greater or equal to 2.74 $\mu$M (Table \@ref(tab:toxallanimal)) and doses effective against their reproduction and mobility at 488.85 nM, or higher (Section \@ref(sublethalsoilworm)). Thus, there is a differential neonicotinoid-susceptibility between *C. elegans* and parasitic nematodes and between *C. elegans* and earth worm species. The same is true among the insects, where some species are much more susceptible than others (Section \@ref(tab:toxallanimal)). Therefore, the sensitivity of each species to neonicotinoids should be considered separately when evaluating the environmental impact of these insecticides. This highlights the complexity and difficulty of the neonicotinoids risk characterisation and usage management. 
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## *C. elegans* as a model to study the mode of action of neonicotinoids

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The adverse effect of neonicotinoids on biological pollinators and the emerging resistance (Section \@ref(resgenevidence)) opposes a threat to the food safety and highlights the need for the synthesis of novel and more selective insecticides. One of the first steps towards the synthesis of new insecticides is the understanding of their mode of action [@metcalf1971]. Neonicotinoids act by targeting insect nAChRs (Section \@ref(neonicstarget)), however their mode of action and receptor specificity differs. They can have agonistic, antagonistic and super-agonistic action, depending on the animal preparation upon which they are applied (Section \@ref(moaneonicsinsects)). In addition, even neonicotinoids sharing the same pharmacophore, target distinct receptors [@thany2009; @moffat2016]. Difficulties in the heterologous expression of insects' receptors hiders their pharmacological characterisation and description of neonicotinoid-receptor specificity (Section \@ref(ric3insect)). 
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Model organism *C. elegans* is an alternative system in which the mode of action of chemical agents can be studied in-vivo [@lewis1987; @lewis1980]. *C. elegans* expresses at least 29 nAChR subunits (Figure \@ref(fig:seqidentityecd-label)) [@jones2007a] which form receptors at the neuromuscular junction and in the nervous system. Muscle-type receptors are involved in the regulation of locomotion, reproduction and feeding (Section \@ref(pharmacelegans) \@ref(nachrutantfeeding)). Neuronal type receptors are expressed in circuits involved in the sensory processing and chemosensation [@yassin2001]. The suitability of the *C. elegans* system for the mode of action studies was investigated by scoring the sensitivity of native pharyngeal nAChRs receptors to neonicotinoids. 
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Pharynx is a neuromuscular system in which feeding is carried out by a musculature under the influence of the pharyngeal nervous system. In the presence of food, 5-HT is released, which stimulates MC neuron. MC releases acetylcholine which acts directly on EAT-2 containing nAChRs to drive fast pumping. Thus, EAT-2 is a molecular determinant of the fast pharyngeal response. Although EAT-2 is the only nAChR subunit with clearly characterised function, ACR-7 is also expressed in the pharyngeal muscle [@saur2013].

To determine the effects of neonicotinoids on the pharyngeal nAChRs, dissected head preparations were exposed to clothianidin, thiacloprid and nitenpyram. Their effects on 5-HT stimulated pharyngeal pumping were investigated. Nitenpyram and clothianidin inhibited 5-HT stimulated pumping at 25 mM and 500 $\mu$M, respectively (Section \@ref(dissectedanimalnicotineandneonics)). The impact of neonicotinoids on unstimulated pharynx was also investigated and revealed no effects of thiacloprid and nitenpyram. In contrast clothianidin stimulated pumping. The lowest dose of clothianidin effective against this *C. elegans* behaviour was 75 $\mu$M. These data suggest nAChR expressed in the pharynx have low affinity to neonicotinoids. In contrast, neonicotinoids are effective at nM concentrations on target insect receptors (section \@ref(electrophysevidence) and \@ref(ligbinding)) suggesting nAChR pharmacophore present in pest and beneficial insects are distinct from those found in the *C. elegans*. 

nAChR pharmacophore consists of the contributions from the $\alpha$ (principal) subunit and non-$\alpha$ (complementary) subunit (Section \@ref(bindingsite) and \@ref(pharmacophore)). Residues important in binding of agonists have been identified in mollousc AChBP and nAChR bound to agonists, including nicotine and acetylcholine, thiacloprid, imidacloprid and clothianidin [@celie2004; @hansen2005; @ihara2008; @talley2008; @ihara2014; @zouridakis2014; @morales-perez2016]. Residues identified as those important in binding of agonists were compared between worm and insect nAChRs, by aligning their sequences. 

Chosen insect subunits are those that form receptor chimeras in the recombinant system and confer high binding affinity to neonicotinoids (i.e Kd below 10 nM, Section \@ref(chimerareceptors) and Table \@ref(tab:bindignrecombinant) and $\beta1$ subunit, identified as a molecular determinant of neonicotinoid-resistance in *M. persicae* [@bass2011]. Amino acid sequences of these subunits were aligned against nAChR subunits forming functional receptors at the body wall muscle, namely ACR-16 [@ballivet1996; @boulin2008; Section \@ref(muscletypenachr)], as well as EAT-2 and ACR-7 which are two subunits identified in the pharyngeal system [@mckay2004; @saur2013 and Section \@ref(nachrinpharynx)].

Comparison of nAChR binding pocket residues in *C. elegans* and insect receptors identified several differences. Basic residue at position 34 in the loop G of the complementary non-$\alpha$ subunit, has been shown to confer high binding affinity of thiacloprid and clothianidin to AChBP [@ihara2014]. No basic residue has been identified in the aligned *C. elegans* sequences. Comparison of the remaining *C. elegans* subunits revealed that 3 have basic residue at that position, namely UNC-63, UNC-38 and ACR-6. UNC-38 and UNC-63 are the $\alpha$ subunits of one of the BWM receptor. There is a binding site formed at the interface of UNC-38 and UNC-63, whereby UNC-63 contributes the complementary residues. Low potency of neonicotinoids in locomotory assays suggest that they bind with low affinity to BWM *C. elegans* receptors, suggesting basic residue in loop G is not the sole determinant of neonicotinoids binding selectivity. 

Sequence alignment of insect and *C. elegans* subunits, revealed further differences including residue corresponding to Gln55 in loop D of AChBP. Basic residue at that has been identified as important in conferring neonicotinoid-susceptibility in structural studies of neonicotinoid and nicotine bound AChBP [@ihara2008; @talley2008] and in genetic studies of insects resistant to neonicotinoids [@hirata2015; @hirata2017; @bass2011]. This basic residue corresponds to neutral or positive residue in most receptors subunits in *C. elegans*.  Out of 5 aligned *C. elegans* sequences, only 1 had basic residue at that position. An alignment of all *C. elegans* subunit against the insect $\beta1$ was carried. Out of 29 subunits, 6 namely ACR-7, ACR-9, ACR-25, ACR-15, ACR-10 an UNC-38 had basic residue at that position (data not shown). 

There are also differences in the loop B amino acid at position 145, which has been identified in the genetic analysis of imidacloprid-resistant strain of *Nilaparvata lugens* [@liu2005] and in loop E which contributes aromatic amino acids to the binding site. 

Taken together, a small number of *C. elegans* nAChR subunits have amino acids corresponding to those identified as those important in conferring neonicotinoid-sensitivity of insects. Despite this, *C. elegans* is not sensitive to neonicotinids, suggesting *C. elegans* nAChRs are of low affinity to these compounds. This suggests that there are other determinants of neonicotinoid-sensitivity, or that multiple residues must be found in a single receptor to confer high affinity to neonicotinoids. 

The differences between the insect and *C.elegans* binding pocket are mainly found in the complementary site, suggesting the contributions from the complementary site determine neonicotinoids-selectivity. This is supported by the literature [@marotta2014; @hansen2004]. For example, swapping of $\beta$ subunits in $\alpha4\beta2$ mammalian receptor diminished cytisine activity on this receptor [@harpsoe2013]. Receptor becomes responsive to cytisine at sub-$\mu$M concentrations by a mutation in a single amino acid in the complementary binding pocket [@marotta2014]. Thus the variation in the complementary binding pocket residues gives rise to ligand binding specifities and pharmacological differences between various compounds and receptors. 
(ref:gendiscussion-celegansinsectalagnment) **Sequence alignment of the pharmacophore of insect and *C. elegans* nAChR subunits.** Ligand binding pocket is formed from the loops originating from the principal (a) and complementary (b) receptor subunits. Amino acids important in forming drug-receptor interactions are color-coded as in Figure \@ref(fig:binding-pocket-label). Non-conserved residues are underlined. Numbering is according to the AChBP of Ls sequence. Mp = *M. persicae* (peach aphid), Dm = *D. melanogaster* (fruit fly), Ce = *C. elegans*. Sequence alignment generated by MUSCLE.

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```{r gendiscussion-celegansinsectalagnment-label, fig.cap="(ref:gendiscussion-celegansinsectalagnment)", fig.scap= "Sequence alignment of the pharmacophore of insect and \\textit{C. elegans} nAChR subunits", fig.align='center', out.width= '80%', echo=FALSE}
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  knitr::include_graphics("fig/gen_discussion/celegans_and_insect_added_loopG_and_B.png")
```


<!-- Lack of sensitivity of neonicotinoids on exposed pharynx suggest that residing nAChs are pharmacologically distinct from those found in insects.  -->
<!-- Conversy, anthelmintic levamisole has potent action on *C. elegans*, but is a weak agonist on insect receptors. Levamisole at conc induce depolarisation of ...*C. elegans* receptors (ref). In contrast, they weakly depolarise cocroach motor neuro [@pinnock1988]. These data suggest that there is a striking difference between the neonicotinoid affinity on insect neurons and on *C. elegans* pharynx.  -->
<!-- Other pharma differences : read this papar: tornoe1994.  -->

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## *C. elegans* pharynx as a platform for the pharmacological characterisation of nAChRs 
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Low sensitivity of *C. elegans* to nitenpyram, clothianidin and thiacloprid in behavioural and cellular assays precludes its use as a model to study the mode of action of neonicotinoids per se, but highlights its potential use as a suitable background for the heterologous expression of insect nAChRs. New insecticides are needed to prevent the negative environmental impact of neonicotinoids and combat emerging resistance. The first step towards the synthesis of neonicotinoids with improved selective toxicity profile is the heterologous expression of nAchRs from pests and non-target species in a suitable host.

Cell lines and Xenopus oocytes are routinely used as biological systems for heterologous nAChR expression (Section \@ref(biologicalsystemfornachrexpression)). Model organism *C. elegans* is an alternative system in  which recombinant proteins can be expressed by generation of transgenic worms [@crisford2011; @sloan2015]. In comparison to other systems, *C. elegans* is cheap and easy to maintain, whereas transgenic worms can be preserved for years at - 80 &deg;C. The function of recombinant nAChRs can be studied in-vivo by evaluating their impact on behaviours underpinned by the cholinergic neurotransmission. 

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Acetylcholine is a major neurotransmitter of the pharynx, released by at least 7 out of 14 neurons (Table \@ref(tab:pharynx-neurons)). It is key and necessary for the induction of fast feeding in response to food (Section \@ref(achpumping)). Its action on the pharynx is mediated by nAChRs, most notably EAT-2 expressed at the NMJ (Section \@ref(nachrinpharynx)). *C. elegans* pharynx does not posses an innate susceptibility to low concentrations of neonicotinoids, creating an appropriate genetic background in which effects of these compounds on recombinantly expressed receptor can be studied. In addition, it expresses an array of chaperon proteins, creating a favorable environment for the maturation and function of these ion channels (Section \@ref(cematnachr)). *C. elegans* pharynx has been successfully used for the heterologous expression of non-native proteins [@crisford2011], including nAChRs [@sloan2015]. 

EAT-2 is a single nAChR subunit that confers pharyngeal 5-HT sensitivity and feeding response in *C. elegans* [@mckay2004]. We show that *eat-2* mutant is a suitable genetic background, in which the expression of heterologous nAChRs could be scored in behavioural assays. Transgenic line in which *eat-2* is heterologously expressed, rescued the blunted pharyngeal response to food and restored 5-HT resistance of the *eat-2* mutant (Section \@ref(behaviourofeat2rescue)). However, the expression of human $\alpha7$ nAChR in the *C. elegans* mutant pharynx had no observable phenotypical consequences (Section \@ref(feedingalpha7celegans) and Section \@ref(htandtransgenicalpha7)). Similarly, expression of this receptor in the wild-type worm revealed no differences in pharyngeal responses to nAChR agonists nicotine or choline (Section \@ref(pharmaalpha7transegnicworms)). Staining with fluorescently labelled human $\alpha7$ nAChR antagonist $\alpha-Bgtx$, reveled increased fluorescence in the pharyngeal muscle of transgenic, when compared to control worms (Section \@ref(bgtxstaining)). $\alpha$-bgtx binds to the extracellular, domain of the receptor [@dellisanti2007]. This suggests that $\alpha7$ receptor is expressed on the cell surface of the pharyngeal muscle, however due to the lack of phenotype its functionality is unclear. Further pharmacological experiments of transgenic strains (as described in the Discussion of Chapter 6) should be carried out to determine whether $\alpha7$ retains its function upon expression in the pharynx of *C. elegans*. 
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The expression of $\alpha7$ was driven a myo-2 promoter, which should drive the expression in all cells of the pharyngeal musculature [@altun2009a]. However, $\alpha-Bgtx$ staining of transgenic worms was concentrated in pm7 and pm8 muscle cells, suggesting $\alpha7$ is expressed in the terminal bulb, which does not overlap with the localisation of native EAT-2, native protein. Thus, native EAT-2 promoter should be used to ensure correct localisation of the expressed protein. 
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<!-- In dissected animal, in the absence of food pharynx continues to pump. It is myogenic or neurogenic?  -->

<!-- Rhythmical pattern of muscle contraction and relaxation can be controlled by neuronal input or the intristic ability of the muscle to contract without the input of the nervous system. Rhythmic contraction of muscle can be driven by the neuoronal network controls the pattern of contraction-relaxation or myogenic if this cycle is control by the intrinsic ability of muscle cells to inititate and control APs without the neuronal input. Laser ablation of all pharyngeal neurons does not abolish pumping entirely suggesting it can act without neuornal input. However In C. elegans, the contraction of pharynx is mostly controlled by ACh. Mutants deficient in production of ACh or ACh neurotrasmission hatch but do not feed and die shortly after hatching [@rand1989, @alfonso1993). Same happens when all neurotrasnsmission is abolished [@nonet1998). In addition, in transgenic adult worms pharmacological switching off of the pharyngeal nervous system abolished pumping entirely [@trojanowski). This supports the neurogenic control of pumping. @trojanowski2016 suggests tonically released ACh from the pharyngeal nervous system drives the myogenic activity of the muscle.  -->

<!-- note that It is also possible that it is controlled by the extrapharyngeal nervous system and humoral neurotransmission.  -->

<!-- Laser ablation of pharyngeal nervous system did not ablate pumping because there were still inputs from the extrapharyngeal nervous system. Supports the idea finding that the pumping rate on dissected animal are lower than those off food on plate (you can show data here) or 5-HT stimulated in intact vs dissected animal. There are two points of conection: between RIP neurons and I1 neurons which are connected by a pair of gap junctions [@albertson1976). However ablation of RIP  -->
<!-- does not led to marked behavioural changes. It did not cause changes in pumping rates on or off food (our lab, data not published, nicos thesis). The exception is the absence of the inhibitory effect on pharynx due to light touch [@riddle1997). However the pharynx can be influenced by extrapharyngeal system. For example, inhibitory responses on pumping of drugs is diminished when a head in cut away from the rest of the body or when I1 neurons are ablated the sensitivity decreases [@dent2000). Also acute inhibitory response to light. Light detected by RIP (or other neurons which synapse onto RIP) neurons signal to I1 which synapses to MC to inhibit pumping. Response is blunted by RIP and I1 ablation [@bhatla2015).  -->



<!-- As mentioned in cut head section, these contraction were weak. Note that these look exactly like the once seen in this paper here: https://www.nature.com/articles/srep22940. Long EPG, small amplitude and long duration. In this paper they stimulated the muscle but the nervosus system is silenced. Hence these effects could be due to a direct effect of nicotine on muscle?  -->

<!-- Some report that bees prefenrentialy feed on food containing thiamethoxam and clothianidin not by sensory processed. thiamethoxam, imidacloprid and clothianidin inhibit B. terrestris feeding, thiamethoxam and clothianidin of A. mellifera [@kessler2015). Bombus terrestris reduction of feeding due to imidacloprid and clothianidin. Thiamethoxame has no effect [@thompson2015). It might be they are either incapable of feedin or choose not to feed. However, @kessler2015 showed they do not act on gustatory or sucrose sensing neurons [@kessler2015). Authors suggest honey bees and bumbkebees cannot taste neonicotinoidss. The exact mechanism of these alteratiosn is to be investigated.  -->

<!-- ###Senory effects of nicotine -->

<!-- Effects of nicotine on food-stimulated pumping was scored by placing worms on palates containing food patch and nicotine incorporated into the agar. Worms escaped the experimental plates containing nicotine at concentrations $\ge$ 25 mM by crawling to the edge of the plate. This suggests nicotine has sensory effect on worms and induces avoidance response. This behaviour was also triggered by nicotine-related compoud quinine [@hilliard2004). There are two potential cellular targets of nicotine in worms: sensory IL1 and IL2. -->
<!-- Cholienrgic IL2 [@pereira2015) labial sensory neurons are present around the mouth of the worm. They are not exposed to the external environment, but are in close proximity to the surface of the worm. IL2 express nAChR DES-2 subunit [@treinin1998). 2 out of 6 IL2 neurons output onto RIP neurons [@Serrano-Saiz2013) which connect the somatic and pharyngeal nervous system [@albertson1976).  -->
<!-- IL1 neurons are neurons positioned around the mounth of the worm and exposed to the external environment. They are involved in mechanosensation [@Kindt2001) and make connections with RIP [@albertson1976). There is some evidence that IL2 express ACR-2 nAChR subunits [@Nurrish1998, @Hallam2000).  -->
<!-- Nicotine can also bind to receptors other than nAChRs, for example TRP channels [@Liu2004, @Talavera2009). TRPV channels are expressed on IL1 neurons [@Kindt2001) and are involved in responses to nicotine in a worm [@feng2006).  -->

<!-- # general discussion -->
<!-- Moreover, *C. elegans* physiology means there is a dual route of exposure potentially to much higher than average concentrations of insecticides. Worms are mobile in soil, therefore can forage in close proximity or make a physical contact with a coated seed, whereas their feeding activity means they are not only making a direct contact with a substance, but also ingesting contaminated material. -->

<!-- Reported doses effective against worms are several orders of magnitude higher than those found in the field [@franciscosanchez-bayo2016]. This might suggest that worms are not impacted by field realistic levels of neonicotinoids in the soil. However, a long term exposure assays should be performed to asses if prolonged exposure leads to bioaccumulation and exacerbate effects.  -->

<!-- look at this article to see the expression levels change in response to chronic nicotine treatment of naive worms https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385460/ -->
<!-- this paper shows that effects of nicotine could be due to receptor containing lev-8 https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1471-4159.2004.02951.x -->
<!-- eat-2 is sensitive to nicotine. eat-18 is not https://pdfs.semanticscholar.org/8df9/6213dbc75e8ae0eb67b391e04fab38ab3903.pdf -->





<!-- In contrary, no potency difference was observed in acute exposure experiment and effects on pumping. The reason may lay in the differential routes of drugs’ entry in these assays. Whilst in liquid, worms generally do not pump [@gomez-amaro2015] hence in acute motility assays drugs must cross the cuticle to reach molecular targets. In contrast, in pumping assays pumping is instigated by addition of 5-HT. As a result, drugs gain access through ingestion and diffusion across the cuticle, equilibrating much more readily.  -->
<!-- Increased potency of compounds was also observed in moderate (24-hour) exposure assays. Thiacloprid and clothianidin had no effect on N2 wild-type worm, but inhibited feeding and egg-laying locomotion of bus-17 mutant at low mM concentrations.  -->


<!-- ###	Neonicotinoids exhibit differential effect on worm behaviour   -->
<!-- In acute and moderate exposure assays, intact worms were subject to treatment and as shown, the cuticle had a major effect on the efficacy of compounds. To investigate the issue of permeability, further assays were employed in the following chapter. circumvent the issue of permeability dissected worm assay was employed. In this assay, C. elegans pharynx is cut away from the rest of the body. By doing so, the cuticular barrier is diminished, whereas drugs only need to cross the basal membrane separating pharynx and external environment to bind to the molecular targets. Isolated pharynx pumps <5 pumps/seconds. By direct application of drugs, their excitatory effects can be tested. In addition, application of 5-HT to elicit pumping and subsequent application of a drug enables determination of inhibitory effect of compounds on 5-HT stimulated pharyngeal pumping. -->

<!-- In general, results from the cut head experiment confirm that the removal of the cuticle resulted in increased efficacy of drugs when compared to the results from the pharyngeal pumping acute exposure assays. The greatest 3 orders of magnitude shift was observed for nicotine. The efficacy of nitenpyram increased 18-fold shift, whereas thiacloprid 3-fold. Clothianidin had no effect in both preparations. Relative shifts in potency suggest neonicotinoids penetrate biomembranes much less readily in comparison to nicotine at least in part due to the physicochemical properties. Generally, nicotine is more hydrophobic and less lipophilic that neonicotinoids and protonated at physiological pH (Table 1.1).  -->

<!-- In exposed pharynx experiments neonicotinoids have shown a differential effect on the pharyngeal pumping. Clothianidin induced a transient excitation. This effect was also observed in honey bee neuronal preparation [@palmer2013]. Thiacloprid induced sustained excitation [@moffat2016] have shown that N-nitroguanidines clothianidin, thiamethoxam and imidacloprid have divergent effect on the behaviour and the development of bumblebees, as well as on the function of mushroom body Kenyon cells. This suggests that neonicotinoids act on different receptors or they may have a differential mode of action in bee and C. elegans. Indeed, clothianidin has been shown to act on both imidacloprid-sensitive and insensitive nAChRs in cockroach isolated SUM ganglion [@thany2009].  -->

<!-- 5-HT stimulates both. Pumping instigated by the action of 5-HT on MC neurons by acting on ser-7 receptors, whereas insthmus peristalsis by the action on M4 neurons by ser-7 too [@song2013a]. Ser 7 are serotonin gated GPCRs [@hobson2003].  -->
<!-- MC and M4 are cholinergic and output directly onTO the muscle. In response to 5-HT, MC and M4 release ACh to stimulate pumping. -->
<!-- >>>>>>> a966d072d44f70c03380ef6b978ec19ddb9bf549 -->

<!-- Sensory stimulation of the pharyngeal system -->



<!--  Hence function might be redundant.  -->

<!-- Ablation of I1 had no effect in the presence of food [@raizen1995), but reduced pumping in the absence of food [@trojanowski2014). I2 is the only cholinergic neuron that does not synapse onto the pharyngeal muscle directly. it activates the pharynx via MC and M2 [@albertson1976).  -->

<!-- SENSORY RESPONSE TO ODOUR  -->
<!-- <<<<<<< HEAD -->
<!-- potentiation of pumping or depression in response to attractive or repelling odourants. Detected by 2 different sets of neurons : AWC and ASH neurons [@li2012).  -->

<!-- note that peristalsis is also regulated by other neurotransmitters. Here is shows that neuroipeptides and monoamines control insthus persistalsis in response to 5-HT  -->
<!-- ======= -->
<!-- potentiation of pumping or depression in  -->

<!-- Anatomically, it is mailny composed from muscle and nerve cells. There are 20 muscle cells . Muscle cells are in the syncytium, so that the electrical signal can be spread almost instantenously from one section of the pharynx to the next. Moreover, the AP can also readily travel from one anatomical structure to the next. That is why the contraction of the corpus and the terminal bulb begin simultaneouly [@raizen1994). -->
<!-- >>>>>>> a966d072d44f70c03380ef6b978ec19ddb9bf549 -->


<!-- <<<<<<< HEAD -->
<!-- Pharynx is a feeding organ of the worm composed of three distinct anatomical features: most anterior corpus, middle isthmus and posterior terminal bulb. Pharynx is located in the head of the worm, separated from the intestine by the and surround by... Its functions it to trap, filter, smash and pass the bacteria to the gut for digestion. These are performed by two motions: pumping and peristalsis. Pumping is the contraction and a susequent relaxation of the corpus, anterior isthmus and the terminal bulb. Peristalsis of the posterior isthus. Anatomically, it is mailny composed from muscle and nerve cells. There are 20 muscle cells [@albertson1976). Muscle cells are in the syncytium, so that the electrical signal can be spread almost instantenously from one section of the pharynx to the next. Moreover, the AP can also readily travel from one anatomical structure to the next. That is why the contraction of the corpus and the terminal bulb begin simultaneouly [@raizen1994). The action of the pharynx is under the control of the pharyngeal system.  -->
<!-- There are 20 neurons of 14 types in the pharynx [@albertson1976), but only three of those have identified as sufficient to elicit feeding. Those are MC which inititiates the pump [@raizen1994, @raizen1995), M3 ends it [@avery1993) whereas M4 is initiating peristalsis [@avery1987).  -->
<!-- The activity of the pharynx is mainly reulated by acetylcholine, glutamine and serotonin.  -->
<!-- Pharyngeal system is connected to the extra-pharyngeal nervous system at a single point. Extrapharyngeal RIP neurons and pharyngeal I1 neurons are connected by a pair of gap junctions [@albertson1976). Sensory resonses modulate pumping. But are not required for normal/intrinsic pumping. Hence laser ablation of RIP has no effect. But it does get rid of the ability to normally respond to the environment. -->

<!-- The presence of familial food is detected by dendrites of ADF head chemosensory neurons situated outiside the pharynx [@sze2000]. A release of serotonin from ADF neurons [@bargmann1991] activates neurosecretory NSM neurons within the pharyngeal nervous system. It has been suggested that NSM sense bacteria in the pharynx lumen as well.  Evidence that stimulation of NSM is more imp in locomotion control than in the pharyngeal control [@cunningham2012]. NSM release 5-HT to the pseudocoelomic fluid to inhibit locomotion and stimulate pumping and alter other bahaviours. Pumping stimulation is due to the action of 5-HT on MC and M3 and M4.  -->
<!-- 5-HT stimulates both. Pumping instigated by the action of 5-HT on MC neurons by acting on ser-7 receptors, whereas insthmus peristalsis by the action on M4 neurons by ser-7 too [@song2013a]. Ser 7 are serotonin gated GPCRs [@hobson2003]. Duration of the action potential is regulated by M3 neurons which release glutamine and causes repolarisation. -->
<!-- MC and M4 are cholinergic and output directly onTO the muscle. In response to 5-HT, MC and M4 release ACh to stimulate pumping.  -->
<!-- Evidence suggests exhogenous 5-HT acts by the enhancement of M3 which releases glutamine to reduce the pump latency and enable fast pumping frequency of ~250 pumps/min.  -->

## *C. elegans* as a model for mammalian toxicity studies 

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To better understand the pharmacological profile of *C. elegans* nAChRs, the effects of endogenous neurotransmitter acetylcholine as well as agonist nicotine and cytisine were tested on the pharynx. Acetylcholine, nicotine and cytisine were applied on cut-head and the effects on EPG was assayed. All three induced potent and transient stimulation of pumping leading to muscle tetanus (Figure \@ref(fig:epg-nicotine-2-label))). Concentrations effective were in the low $\mu$M range. The order of potency as measured by the EC~50~ was: nicotine > cytisine > acetylcholine. In comparison to human $\alpha7$, effective concentrations were in a similar range [@papke2002]. In addition, like pharyngeal nAChRs, human $\alpha7$ receptors insensitive to low doses of neonicotinoids with the EC~50~ values of 0.74 mM and 0.73 mM, respectively for clothianidin and imidacloprid, on the heterologously expressed channel [@cartereau2018]. This suggests the pharmacophore of human $\alpha7$ and pharyngeal nAChRs is conserved. This was confirmed by aligning the sequences of key amino acids forming the nAChR binding pocket in $\alpha7$ and two of the pharyngeal nAChRs (Figure \@ref(fig:pharmacophoreceleganspharynxandhumanalpha7-label)). 
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(ref:pharmacophoreceleganspharynxandhumanalpha7) **Amino acid sequence alignment of human $\alpha7$ and two of the pharyngeal *C. elegans* nAChRs ligand binding pockets.** Amino acid sequences forming nAChR binding pocket were aligned. Amino acids important in agonist binding are highlighted, as in Figure \@ref(fig:binding-pocket-label). Ce = *C. elegans*, Hs = human. Sequence alignment generated with MUSCLE.
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```{r pharmacophoreceleganspharynxandhumanalpha7-label, fig.cap="(ref:pharmacophoreceleganspharynxandhumanalpha7)", fig.scap="Amino acid sequence alignemnt of human $\\alpha7$ and pharyngeal \\textit{C. elegans} nAChRs ligand binding pockets.", fig.align='center', out.width= '150%', echo=FALSE}
knitr::include_graphics("fig/gen_discussion/celeganseat2andacr7andhumanalpha7.png")
```

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Comparison of human $\alpha7$ and pharyngeal nAChR subunits EAT-2 and ACR-7 revealed high sequence similarity. Almost all residues forming ligand binding pocket are conserved between these subunits, suggesting pharyngeal nAChR subunits are homologous to human $\alpha7$. Besides pharyngeal nAChR subunits, homologs of over two thirds of human proteins can be found in *C. elegans* [@sonnhammer1997; @lai2000]. The similarities between mammals and *C. elegans* extends beyond genetics. *C. elegans* has conserved synaptic function, due to the presence of almost all vertebrate neurotransmitters and conserved neuronal signalling pathways [@bargmann1998; @kaletta2006]. There are however some differences. *C. elegans* presents expresses inhibitory glutamate-gated chloride channel absent in mammals [@cully1994], but lacks voltage-gated sodium channels which is present in humans [@bargmann1998]. Despite these limitations, *C. elegans* emerges as an attractive model for toxicity studies [@hunt2017]. 
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Methods to use *C. elegans* as a model to study acute toxicity as well as developmental and reproductive toxicology have been developed [@boyd2010; @xiong2017]. A good correlation between the rank order of toxicity of many compounds on *C. elegans* and mammals for acute toxicity, growth and reproduction endpoints have been found [@williams1988b; @boyd2010]. This includes organophosphates, which act by disrupting cholinergic neurotransmission at the synapse [@chadwick1947]. The rank order of acute toxicity, as measured by LC50, correlated well with the LD50 ranking of these agents in rats and mice [@cole2004]. This highlight the potential suitability of *C. elegans* as a model for toxicity testing of cholinergic and other neurotoxins. Introduction of *C. elegans* into toxicity testing has a potential to reduce the use of conventional mammalian models, resulting in the reduction of cost and duration of such studies [@williams1988].
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<!-- ## Cholinergic drive of the *C. elegans* pharynx -->

<!-- Cholinergic neurotransmission is crucial in driving the food evoked pharyngeal response in *C. elegans*. Experiments were carried out to investigate the cholinergic drive of the *C. elegans* pharynx. In the presence of food, wild-type *C. elegans* pumps at a rate of 4.5 Hz. This effect can be mimicked by the application of 5-HT. In the presence of 5-HT, pharyngeal pumping of dissected *C. elegans* increases with the EC~50~ of 169 nM. This supports the 5-HT driven stimulation of pumping, whereby in the presence of food, 5-HT is released from pharyngeal MC neuron to stimulate pumping. It was also shown that the 5-HT driven and food driven pharyngeal pumping is dependent on EAT-2 nAChR. However, high rates of pharyngeal pumping can be induced with nicotine and clothianidin in both wild-type and *eat-2* mutant. This suggests there is an alternative, EAT-2 independent pathway inducing fast pumping in *C. elegans*. This pathway could involve other nAChRs because it is likely that there are other receptors expressed in the pharyngeal muscle. The expression pattern of nAChR auxilary subunit EAT-18 is much more diffused than that of EAT-2 receptors. EAT-2 maps onto pm4 and pm5 muscle, whereas EAT-18 on all pharyngeal muscles and M5 neuron [@mckay2004]. The identity of these receptors, (with the exception of ACR-7 of unknown function) is to be determined. -->

<!-- Nicotine and clothianidin may act at least partially independently on the MC.  Stimulation of MC neurons in *eat-2* mutant (in the presence of metabotropic ACh antagonist) also elicits pumping response, but only to 0.7 Hz [@trojanowski2014]. In the presence of nicotine and clothianidin pharynx of the *eat-2 C. elegans* strain pump at a maximum measured average rate of 2.1 and 4.2 Hz, suggesting both compounds by-pass MC.  -->

<!-- <!-- Nicotine may act to elicit potent pharyngeal response by proteins other than nAChRs. The pharyngeal response to nicotine in *eat-18* mutant was reduced, not diminished [@raizen1995]. However this could also mean that there are nAChRs capable of functioning independetly of EAT-18.  --> 

<!-- <!-- Response of the pharynx to clothianidin are distinct from classical nAChR agonist responses, therefore it is also possible that it acts on proteins other than nAChR to stimuate pharynx.  --> 

<!-- Therefore, this study suggests that there are multiple routes for the activation of the *C. elegans* pharynx. MC - EAT-2 pathway is involved in food and 5-HT driven response, whereas nicotine and clothianidin may elicit pumping via an independent pathway(s), which remains to be identified. -->


<!-- (fig:seq-sim) **The sequence similarity between the ECD of *C. elegans*, insect and human receptors.** The sequence similarity between amino acid sequences computed with -->

<!-- ```{r seq-sim-label, fig.cap="(fig:seq-sim)", fig.scap='The sequence similarity between the ECD of \\textit{C. elegans}, insect and human receptors.', fig.align='center', echo=FALSE} -->

<!-- knitr::include_graphics("fig/results3/similarity_pharyngeal_am_hs.png") -->
<!-- ``` -->

<!-- (fig:seq-iden) **The sequence identity between the ECD of *C. elegans*, insect and human receptors.** -->

<!-- ```{r seq-iden-label, fig.cap="(fig:seq-iden)", fig.scap = 'The sequence identity between the ECD of \\textit{C. elegans}, insect and human receptors.', fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results3/identity_pharyngeal_am_hs.png") -->
<!-- ``` -->