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\title{\LARGE {\bf Investigation of the selective toxicity of neonicotinoids using the nematode worm Caenorhabditis elegans}\\
\title{\LARGE \textbf {Investigation of the selective toxicity of neonicotinoids using the nematode worm \textit {\textbf {Caenorhabditis elegans}}}\\
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\author{Monika Kudelska}
\maketitle
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......@@ -41,7 +41,7 @@ library(kableExtra)
In 1970s, the scientists of Shell Development Company Biological Research Centre in California identified alpha- DBPN (2-(dibromonitromethyl)-3-(methylpyridine)), first synthesised by Prof. Henry Feuer [@feuer1986]. This lead compound showed low insecticidal activity against aphid and house fly [@tomizawa2003; @tomizawa2005]. Structural alterations of DBPN resulted in production of nithiazine (Figure \@ref(fig:neonics-structure-label)). Nithiazine showed improved insecticidal activity and was particularly effective as a new housefly repellent [@kollmeyer1999]. Further replacement of the thiazine ring by chloropyridinylmethyl (CPM) group, addition of the imidazolidine or its acyclic counterpart, and retention of the nitromethylene group resulted in generation of more potent compounds, one of which, nitenpyram, exhibited particularly high efficacy. Regrettably, both nithiazine and nitenpyram are not useful in fields, as they are unstable in light. The latter however is successfully used in veterinary medicine as an external parasite treatment for cats and dogs.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(fig:neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
Research into novel neonicotinoids continues [@shao2013]. In the last decade, several novel insecticides have been characterised and approved for use in the EU. Sulfoxafrol [@zhu2011; @eu2019a] and flupyradifurone [@nauen2015; @eu2019b] have been classified as representatives of new chemical classes, namely sulfoximines and butenolides. However, due to their mode of action and similar biochemical properties, some argue that they are in fact neonicotinoids, whereas their mis-classification has been deliberate to avoid association with neonicotinoids [@pan2019].
......@@ -348,7 +348,7 @@ The effects of neonicotinoids on the neuronal transmission was investigated on i
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects. This conclusion was supported by the behavioural observation, whereby the neonicotinoid intoxication mirrors intoxication seen with cholinergic agents (Section . In response to imidacloprid, insects become hyper excited as evident by excessive pacing. They then collapse and exhibit diminishing uncoordinated leg and abdomen movement until eventual death [@sone1994; @elbart1997; @suchail2001]. Sub-lethal doses (i.e. < 4 nM) have distinct effect, such as an inhibition of feeding leading to starvation [@nauen1995; @elbart1997].
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured cocroach [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons. These data provide evidence that neonicotinoids act directly on the post-synaptic neuron in both target and non-target insects.
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membranes in the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated thoracic ganglion [@tan2008], terminal abdominal ganglion of the americal cocroach [@ihara2006] and in the honeybee [@palmer2013] and fruit fly [@brown2006] neurons.
Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC50 values (concentrations at which the half of the maximum current was observed) are in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(potentpests)). Neonicotinoid-induced currents were reminiscent of those induced by acetylcholine and nicotine, and were prevented by the application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine), suggesting neonicotinoid-induced currents are due to the activation of nicotinic receptors.
......@@ -586,7 +586,7 @@ Recombinant insect nAChR are notoriously difficult to express. Several intervent
## General biology ##{#genbiology}
*C. elegans* exists as a male and hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9 % of worms are hermaphrodites, which self-fertilize their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15^o^C, it takes 5.5 days from egg-fertilization to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25 deg;C, respectively (Figure \@ref(fig:life-cycle-label)). At 20 degrees, hermaphrodite lay eggs 2.5 hours after the fertilisation. 8 hours later the embryo hatches as a larvae in the first stage of its development (L1). In the presence of food, larvae develops into an adult through three further developmental stages, namely L2, L3 and L4. The transition between each larval stage is marked by a process of maulting, during which the old cuticle is shed and replaced by a new one. In the absence of food, developing L2 and L3 worms enter the dauer stage. The worms can remain arrested at this low metabolic activity state for up to several weeks, and will develop into adults, should the food re-appear. Hermaphrodites remain fertile for the first three days of their adulthood. Their eggs can be fertilised internally with the sperm produced by the hermaphrodite, or, if there are males available, by mating. Unmated worm can lay up to 350 eggs, whereas mated over a 1000 eggs. Figure \@ref(fig:life-cycle-label) illustrated the full *C. elegans* life cycle.
*C. elegans* exists as a male and hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9 % of worms are hermaphrodites, which self-fertilize their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15^o^C, it takes 5.5 days from egg-fertilization to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25 $^\circ$C, respectively (Figure \@ref(fig:life-cycle-label)). At 20 degrees, hermaphrodite lay eggs 2.5 hours after the fertilisation. 8 hours later the embryo hatches as a larvae in the first stage of its development (L1). In the presence of food, larvae develops into an adult through three further developmental stages, namely L2, L3 and L4. The transition between each larval stage is marked by a process of maulting, during which the old cuticle is shed and replaced by a new one. In the absence of food, developing L2 and L3 worms enter the dauer stage. The worms can remain arrested at this low metabolic activity state for up to several weeks, and will develop into adults, should the food re-appear. Hermaphrodites remain fertile for the first three days of their adulthood. Their eggs can be fertilised internally with the sperm produced by the hermaphrodite, or, if there are males available, by mating. Unmated worm can lay up to 350 eggs, whereas mated over a 1000 eggs. Figure \@ref(fig:life-cycle-label) illustrated the full *C. elegans* life cycle.
(ref:life-cycle) **The life cycle of *C. elegans*.** *C. elegans* develops into an adult through 4 larval stages L1- L4. These stages are separated by molts associated with shedding of an old and exposure of a new cuticle. Adults emerge can lay over a 1000 eggs a day which hatch within several hours. Dauer stage is a metabolic compromised worm stage entered in the absence of food. Upon re-appearance of food, worms develop into L4 and adults normally. Figure taken from www.wormatlas.org.
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......@@ -196,7 +196,6 @@ Clothianidin and thiacloprid at concentrations $\ge$ than 1.2 $\mu$M and the EC5
##### Soil nematodes #####{soilnematodesneonicstoxicity}
Neonicotinoids also induce a sublethal effect on the the free-living nematode *C. elegans*. Thiacloprid and imidacloprid have an effect on the reproduction of *C. elegans* with EC50 of 1.14 nM and 2.09 mM, respectively [@gomez-eyles2009]. Thiacloprid at 37 nM has an effect on chemosensing, whereas at 18 $\mu$M it impairs motility of this free living nematode [@hopewell2017]. Impaired motility of *C. elegans* in response to $\ge$ 120 $\mu$M imidacloprid was also recorded [@mugova2018]. Taken together, neonicotinoids have sublethal effects on earth worms and soil nematodes at concentrations as low as nM.
Most of the doses effective against worms are higher than the average doses of neonicotinoids in the field. However, the presence of clothianidin, imidacloprid and thiamethoxam has been detected at lower than average levels, such as 80.10 nM for imidacloprid, 23.01 nM for imidacloprid and 68.56 nM for thiamethoxam [@jones2014]. This suggests that the environmentally relevant concentrations of neonicotinoids may negatively impact on the the well-being of soil dwellers.
<!-- Data regarding toxicity of neonicotinoids on soil worms and nematodes focuses on earthworm *Eisenia fetida* (*E. fetida*, redworm). The toxicity is assessed either following treatment in solution or in the artificial soils. In solution, the LC50 of imidacloprid after 24 hour exposure is 62.08 $\mu$M [@luo1999]. This increases to 24.24 $\mu$M after 48 hour exposure [@luo1999]. Clothianidin is less potent; its LC50 after 14 days is 24.27 $\mu$M [@decant2010]. -->
......@@ -301,30 +300,41 @@ Genetic studies identified other amino acids with a potential importance in conf
Loop B, D and G originate from the complementary site, but the principal site may also play a role. Studies on Drosophila/chicken $\alpha2\beta2$ hybrid and chicken $\alpha2\beta4$ receptors showed that the presence of nonpolar proline in YXCC motif of loop C enhances affinity, whereas mutation of proline to glutamate markedly reduces affinity of neonicotinoids to these receptors [@shimomura2005]. The importance of C-loop regions was also demonstrated by @meng2015 who showed that chimera receptors are deferentially sensitive to imidacloprid at least partly due to the difference in loop C region, equivalent to Ls184-191.
### Cholinergic neurotransmission
### Cholinergic system in insects
#### Enzymes at the cholinergic synapse
<!-- Upon arrival of an electrical signal at the presynpatic terminal, acetylcholine is released into the synaptic cleft. It then binds to nicotinic acetylcholine receptors (nAChRs) expressed at the post-synpatic membrane. Binding of acetylcholine results in depolarisation and excitation of the post-synaptic neurons, or muscle contraction at the neuromusclular junction (NMJ) [@hille1978]. Acetylcholine can also act on other class of receptors, the metabotropic cholinergic G-protein coupled receptor, which are involved in the modulatation of neurotransmission release. The acetylcholine-evoked signal is terminated mainly by synaptic enzyme cholinesterase which hydrolyses acetylcholine to choline and acetate [@fukuto1990], but also by choline uptake to the presynaptic cell by Na^+^-choline transporter. -->
Cholinergic neurotransmission is the process of signal propagation between neurons as well as neurons and muscle cells mediated by a neurotransmitter acetylcholine (ACh) (Figure \@ref(fig:cholineric-synapse-label)) [@williamson2009].
nAChRs mediate fast synaptic transmission at the cholinergic synapse. Cholinergic neurotransmission is the process of signal propagation between neurons as well as neurons and muscle cells mediated by a neurotransmitter acetylcholine (ACh) [@williamson2009]. Cholinergic synapse is characterised by the presence of several proteins which mediate the breakdown, the synthesis and processing of the neurotransmitter (Figure \@ref(fig:cholineric-synapse-label)).
##### Choline acetyltransferase
Choline acetyltransferase (ChAT) is an enzyme synthesising ACh [@greenspan1980b], by a transfer of acetyl-choA onto choline. There are at least two isoforms in Drosophila, which are produced by alternative splicing from the ChAT gene [@slemmon1982]. One is membrane bound, whereas the other is soluble, both of which are 75 kDa [@pahud1998]. The soluble isoform performs the mojority of enzymatic activity. A 65 kDa soluble isoform of ChAT was also isolated from the Locust *Schistocerca gregaria* [@lutz1988]
##### Vesicular acetylcholine transferase
Vesicular acetylcholine transferase (VAChT) mediates ATP-dependent transport [@varoqui1996], which packs ACh into the synaptic vesicles for release [@song1997]. In Drosophila, a single VAChT gene was identified. It is embeddded within the ChAT gene [@kitamoto1998].
##### Acetylcholinesterase
The function and properties of these receptors were studied using mammalian and amphibian muscle preparations.
Acetylcholinesterase (ACE) is a soluble enzyme that catalyses breakdown of ACh [@chao1980; @hsiao2004]. In Drosophila, it is encoded by the Ace locus [@hall1976]. Acetylcholinesterase is a homodimer covalently bonded by the disulphide bridge [@chao1980; @hsiao2004]. Each monomeric subunits is ~67 kDa, folded into 4-helix bundle [@harel2000].
In 1930s, @brown1936; @bacq1937 demonstrated that the application of acetylcholine, as well as nicotine and choline to the isolated mammalian muscle leads to sustained contraction, as showed by the increase in the muscle tension. The muscle contraction was associated with an increase in the frequency of the action potential firings [@brown1936] and the depolarisation of the end-plate [@katz1957]. Acetylcholine-evoked responses could be inhibited by pre-incubation with several compounds, including snake venom proteins, $\alpha$-bungarotoxin [@chang1963].
<!-- The function and properties of these receptors were studied using mammalian and amphibian muscle preparations. -->
Prolonged exposure to high concentration of agonist has a secondary effects on the muscle: desensitisation [@katz1957]. Desensitisation is a post-contraction period, after the removal of the agonist, at which the muscle is relaxed and a subsequent contraction cannot be elicited [@thesleft1955]. Recovery from desensitisation typically lasts few seconds after the agonist removal [@bouzat2008], however the duration varies depending on the receptor, the compound [@briggs1998] and its concentration [@gerzanich1994]. Full recovery may not occur or may be slower if the receptor are exposed to the large doses of agonist for a prolonged time [@katz1957].
<!-- In 1930s, @brown1936; @bacq1937 demonstrated that the application of acetylcholine, as well as nicotine and choline to the isolated mammalian muscle leads to sustained contraction, as showed by the increase in the muscle tension. The muscle contraction was associated with an increase in the frequency of the action potential firings [@brown1936] and the depolarisation of the end-plate [@katz1957]. Acetylcholine-evoked responses could be inhibited by pre-incubation with several compounds, including snake venom proteins, $\alpha$-bungarotoxin [@chang1963]. -->
Biochemical, electrophysiological, genetic and pharmacological approaches were utilised to identify the role of nAChRs in neurotransmission. nAChRs were solubilised and purified from the electric organ of the *Torpedo* and reconstituted into liposomes [@anholt1982]. In the presence of acetylcholine and other agonists, the ionic current was elicited, confirming nAChR is an ion channel [@anholt1982]. Acetylcholine evoked activation ans desensitisation of nAChRs, which correspond to the effects in the muscle, provided evidence that nAChRs mediate fast synaptic transmission at the cholinergic synapse.
<!-- Prolonged exposure to high concentration of agonist has a secondary effects on the muscle: desensitisation [@katz1957]. Desensitisation is a post-contraction period, after the removal of the agonist, at which the muscle is relaxed and a subsequent contraction cannot be elicited [@thesleft1955]. Recovery from desensitisation typically lasts few seconds after the agonist removal [@bouzat2008], however the duration varies depending on the receptor, the compound [@briggs1998] and its concentration [@gerzanich1994]. Full recovery may not occur or may be slower if the receptor are exposed to the large doses of agonist for a prolonged time [@katz1957]. -->
Kinetic properties of nAChRs were studies with the patch clamp technique. Patch clamp is a technique which enables for the resolution of agonist-evoked responses at a single receptor level [@colquhoun1981; @colquhoun1985]. In response to acetylcholine the channel switches between active and inactive form, with the active form interrupted by the short-lived channel closing bursts. Temporal characterisation revealed the average duration of each event. The receptor remains opened for 1.4 ms; this is interrupted by channel closing bursts of 20 $\mu$s which occur at a frequency of 1.9 closures/opening burst. The mean period between the successive channel openings is 342 ms. Further experiments provided the showed that channel opening is not an all or nothing event. Instead, a channel exhibits multiple conductance states, one on which it is fully opened, named a full conductance state (i.e. the active form), and one in which the channel is partially opened, named the sub-conductance state [@colquhoun1985]. The fine structure of full conductance states (or opening bursts) and sub-conductance states varies depending on the agonist used and a receptor protein [@colquhoun1985; @nagata1996; @nagata1998].
<!-- Biochemical, electrophysiological, genetic and pharmacological approaches were utilised to identify the role of nAChRs in neurotransmission. nAChRs were solubilised and purified from the electric organ of the *Torpedo* and reconstituted into liposomes [@anholt1982]. In the presence of acetylcholine and other agonists, the ionic current was elicited, confirming nAChR is an ion channel [@anholt1982]. Acetylcholine evoked activation ans desensitisation of nAChRs, which correspond to the effects in the muscle, provided evidence that nAChRs mediate fast synaptic transmission at the cholinergic synapse. -->
(ref:cholineric-synapse) **Chemical transmission at the cholinergic synapse.** Upon excitation of the presynaptic neuron (1), synaptic vesicles fuse with the membrane, releasing neurotransmitter (2). Neurotransmitter binds to the ligand-gated ion channel (LGIC) expressed on the post-synaptic membrane (3) leading to opening of ion channels and a flux of ions down their electrochemical gradient (4). This leads to either excitation or inhibition of the post-synaptic neuron (5). The signal is terminated by the action of cholinesterase which cleaves the ACh into choline and acetic acid. Choline is then transported back into the synapse and used to make more ACh.
<!-- Kinetic properties of nAChRs were studies with the patch clamp technique. Patch clamp is a technique which enables for the resolution of agonist-evoked responses at a single receptor level [@colquhoun1981; @colquhoun1985]. In response to acetylcholine the channel switches between active and inactive form, with the active form interrupted by the short-lived channel closing bursts. Temporal characterisation revealed the average duration of each event. The receptor remains opened for 1.4 ms; this is interrupted by channel closing bursts of 20 $\mu$s which occur at a frequency of 1.9 closures/opening burst. The mean period between the successive channel openings is 342 ms. Further experiments provided the showed that channel opening is not an all or nothing event. Instead, a channel exhibits multiple conductance states, one on which it is fully opened, named a full conductance state (i.e. the active form), and one in which the channel is partially opened, named the sub-conductance state [@colquhoun1985]. The fine structure of full conductance states (or opening bursts) and sub-conductance states varies depending on the agonist used and a receptor protein [@colquhoun1985; @nagata1996; @nagata1998]. -->
(ref:cholineric-synapse) **Enzymes and transporters at the cholinergic synapse.** Upon release into the synaptic cleft, acetylcholine is broken down to choline and acetate by acetylcholinesterase (AChE). Choline is taken up to the pre-synapse by a choline transporter (ChT). The acetyl group in transferred onto choline to product acetylcholine; a reaction catalysed by choline transferase (ChAT). Generated acetylcholine is pumped back into the synaptic vesicle by the vesicular acetylcholine transporter (AChT) for re-cycling.
```{r cholineric-synapse-label, fig.cap="(ref:cholineric-synapse)", echo=FALSE, fig.scap= 'Chemical transmission at the cholinergic synapse.', fig.align='center', out.height = '60%', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/synapse_general_2.png")
knitr::include_graphics("fig/general_intro/png/synapse_with_enzymes.png")
```
<!-- This phenomenon is due to the distinct conformation of nAChRs, at in which they are not capable of ion conduction [@nemecz2016]. -->
<!-- In the continual presence of agonist, the membrane potential gradually repolarises until it reaches the resting state, hovewer the next depolarisation will not occur, until the agonist are washed off and the receptor recovers from desensitisation [@katz1957]. -->
<!-- Development of molecular cloning technique enabled for expression and kinetic characterisation of nAChRs. In 1985, @mishina1984 generated DNA constructs containing cDNA sequences encoding for the muscle type nAChR. These sequences were injected into the *Xenopus oocytes* [@mishina1984]. Upon application of acetylcholine, a current was recorded, suggesting successful cell surface expression. Single channel recordings revealed that in the presence of agonist, nAChR channel switches between active and inactive form. The active form comprises short-lived channel closing and opening [@mishina1986] and longer pauses in-between the receptor twitching. In addition, channel opening does not seem to be an all or nothing event. Instead, a channel exhibits multiple conductance states, one on which it is fully opened, named a full conductance state, and on in which the channel is partially opened, names sub-conductance state [@nagata1996; @nagata1998]. -->
......@@ -332,89 +342,213 @@ knitr::include_graphics("fig/general_intro/png/synapse_general_2.png")
<!-- $\alpha$-bungarotoxin ($\alpha$-bgtx), is a 74-amino acid long, 8 kDa proteins isolated from the venom of a snake *Bungarus multicinctus*. It binds with high affinity to the NMJ post-synaptic membranes [@lee1967] and blocks synaptic responses evoked by acetylcholine and other agonists [@chang1963] by blocking the access of an agoinist to the nAChR binging site [@@mishina1984]. -->
## Biological relevance of the cholinergic neurotransmission ##{#insectachtransmission}
<!-- Behavioural analysis of Drosophila mutants in which cholinegic neurotransmission is diminished, provides evidence for the role of cholinergic neurotransmission in insects. Drosophila Ace null mutants are lethal: flies dye in the early development [@hall1976; @greenspan1980]. CAT mutants Lethal null mutation of drosophila choline acetyltransferase lethal flies dye during embryogenesis. some homozygus are viable but only at 18 deg c (temp sensitive) - some are temperature sensitive: viable at 18 deg due to the sufficient enzyme activity which is dimonished when flies placed in 30 deg. Upon sfift to high temp: characterisitc behaviour: loss of coordinated movement leading to paralysis and evenual death [@greenspan1980b] and disrupted cotrtnership behaviour. REDUCED MOTILITY IN vesicular acetylcholine transporter gene (Vacht) mutant [@kitamoto2000]. -->
<!-- It is effective against plant insect pests [], and used in organic farming in form of tobacco tea [@isman2006] -->
<!-- Exposure of insects to lethal dose of nicotine results in symptoms characteristic for the nervous system intoxication [@chadwick1947] and include increased locomotory activity, followed by convulsions, twitching and eventual death [@mcindoo1943]. -->
#### Localisation of cholinergic neurons in insects
Enzymes present at the cholinergic synapse have been used as markers for detection of cholinerguic neurons in insects. (1) Immunocytochemistry with monoclonal antibodies specific to ChAt and ACE, (2) in-situ hybridization using using sequences complementary to the ChAT mRNA (3) colorimetric technique for detection of AChE activity [@karnovsky1964] (4) and reporter gene fused to the ChaT gene regulatory elements outlined the presence of cholinergic pathways in Drosophila [@buchner1986; @gorczyca1987; @barber1989; @yasuyama1999], honeybee [@kreissl1989] and locust *Locusta migratoria* [@lutz1987; @geffard1985]. Based on these data, cholinergic neurons are in almost all regions of the brain, in the peripheral nervous system, viz. visual system and the antenna. They are also present in the thoracic, abdominal and terminal abdominal ganglia involved in the regulation of movement of wings, abdomen and legs, as well as the regulation of the anal and reproductive muscles [@smarandache-wellmann2016].
<!-- . In particular, the muchroom bodies associated with learning, formation of memory and the sensory processing [@heisenberg1998], dorsal lobe where mechanosensory and gustatory neurons project into from the anteanna -->
<!-- In peripheral nervous tissue, cholinergic neurons are the -->
Cholinergic neurons have been also mapped by using radiolabelled ligand, specific for nAChRs. $\alpha$-bungarotoxin ($\alpha$-bgtx), is a 74-amino acid long, 8 kDa proteins isolated from the venom of a snake *Bungarus multicinctus*. It binds with high affinity to nAChR [@lee1967] and blocks synaptic responses evoked by acetylcholine and other agonists [@chang1963] by blocking the access of an agoinist to the nAChR binging site [@mishina1984]. $\alpha$-bgtx staining mapped to brain regions identified by other methods. In the honeybee, it was found in the optic lobes, antennal lobes, ocellar system and muchroom bodies [@scheidler1990]. This correlated with the $\alpha$-bgtx staining distribution in the central nervous system of Drosophila [@schmidt-nielsen1977], moth *Manduca sexta* [@hildebrand1979] and cocroach [@orr1990]. Incubation of $\alpha$-bgtx with the ganglia of the american cocroach [@sattelle1983] and cricket *Acheta domesticus* [@meyer1985] identified further regions with high affinity to this protein. Staining was evident in the abdominal ganglion in the region rich in interneurons which make synaptic connections with the sensory afferent neurons [@daley1988] in the abdominal ganglia of the cocroach and in the thoracic ganglia [@sattelle1981]. Presence of nAChRs at the insect ganglia was confirmed using electrophysiological approaches [@sattelle1981; @bai1992]
### Role of nAChRs in insects
Based on the distribution of cholinergic-synapse markers and the quantitative analysis of acetylcholine in the brain [@florey1963], it was concluded that acetylcholine is a major neurotransmitter in the nervous system of insects. In contrast to vertebrates and *C. elegans*, acetylcholine in insects does not mediate signal transduction at the NMJ, insteed it is mainly involved in the sensory pathways and central information processing. The action of acetylcholine is mediated by nAChRs.
<!-- Lerning: [@kerkut1970] acetylcholinesterase inhibitor facilitated the ability of cocroach to learn to raise its leg out of the solution in response to electrical stimuli. Drugs: neostigmine and physostigmine which inhibit acetylcholinesterase [@carlyle1963]. -->
The biological role of nAChRs in insects was investigated in response to nAChR agonists. Lethal doses of neonicotinoid imidacloprid induces complex symptoms in American cocroach and in honeybee [@sone1994; @elbart1997; @suchail2001]. The following order of events was noted: hyperexcitation as evident by excessive pacing, collapse and diminishing uncoordinated leg and abdomen movement followed by paralysis and eventual death. Lethal dose of insecticide nicotine [@david1953], a naturally occurring alkaloid found in the *Solanaceae* family of plants, including tobacco [@steppuhn2004] induce similar effects on bees [@mcindoo1943]. Distinct behavioural alterations can be induced by nAChR agonists at sub-lethal doses.
Imidacloprid at < 4 nM inhibits feeding of *Myzus persicae*, which leads to their starvation [@nauen1995; @elbart1997]. In non-target species, such as honeybees and bumblebees neonicotinoids impair on learning and memory, as well as reproduction (Section \@ref(sublethalbees)).
### Electrohysiological properties of insect nAChRs
Role of nAChRs in insect cholinergic neurotransmission electrophysiological recordings : electrophysiological recordings from the insect ganglia where sensory neuron makes synpatic connections to an interneuor. Robust preparation, easy to dissect and relatively large.
extracellular recordings from the cricket terminal abdominal ganglion recording of the interneuron activity nicotine - cholienrgic and evidence of nAChr expression from the biochem studies. In response to Iontophoretic nicotine, increase in the the rate of neuronal firing, blocked by bgtx [@meyer1985] evidence that nachr agonist lead to excitation.
In cocroach, cell bodies of the dorsal unpaired median (DUM) neurons in the thoracic ganglion depolarised in the presence of nicotine and was blocked by selective antagonist mecamylamine [@bai1992].
In the abdominal ganglia of the cocroach [@sattelle1981].
Inward, depolarising current in isolated
Intracellular recordings have shown that these compounds depolarise and excite the post synaptic neuron of the terminal abdominal ganglia [@kerkut1969]. However, high concentrations 1 mM - 10 mM were needed, potentially due to the high concentration of acetylcholinesterase which rapidly breaks down ACh.
In cultured cells from honeybee antenal lobe whole patch clamp technique: nicotine depolarising inward current inhibited by bgtx [@barbara2005] cultured cells of mushroom bodies of the honey bee [@goldberg1999; @palmer2013] also inhibited by bgtx. Also cultured CNS neurons of Drosophila [@brown2006].
DESENSITISATION
Exposure to high concentrations of agonist had a secondary effects : in the presence of agonist, the depolarising current slowly decreases until it is abolished due to desensitisation [@goldberg1999]. The time constant for desensitisation varied beteween compounds 390 and 420 ms for nicotine [@goldberg1999] up to 23 s in the cocroach neurons [@salgado2004]
Desensitisation is a period after agonist removal, whereby subsequent depolarisation cannot be elicited by agonist [@goldberg1999]. Recovery is reverseble from desenstisiation is dependes on the compounds -, neuronal preparation and concentration. In cultured cells of the honey bee lasts up to 3 minutes after application of Ach/nicotine [@goldberg1999]. in the isolated thoracic neurons up to 30 s [@salgado2004]. Althout recovery is typically reversable, this does not always happen Full recovery may not occur or may be slower if the receptor are exposed to the large doses of agonist for a prolonged time [@katz1957].
isolated thoracic ganglia of the americal cocroach - desensitising in under a second in the presence of ACh and nicotine
CONDUCTANCE
cultured muschroom bodies of the honey bee. Cationic conductance, mainly sodium and potassium but also calcium [@goldberg1999].
cocroach terminal abdominal ganglion extracellular post synaptic potentials in response to acetylcholine. In the presence of acetylcholinenesterase inhibitor acetylcholine at much lower doses 1 uM - 1 mM elicited Rapid and concentration dependent depolarisation excitation of the post-synapse and the gradual decline of the EPSP leading to their block [@callec1973; @sattelle1976].
SINGLE CHANNEL RECORDING
Complex channel kinetics resembling those found in veretbrates [@colquhoun1985; @nagata1996; @nagata1998].
from cholinergic neurons of the larva Drosphila CNS [@albert1993; @brown2006], Musca domestica cultured CNS neurons [@albert1993]
Single channel recordings
In response to acetylcholine the channel switches between active and inactive form, with the active form interrupted by the short-lived channel closing bursts. Temporal characterisation of these events revelaved that the frequency of channel opening and the duration of opening differs depending on the agonist applied and the neuronal preparation. However, typically receptor remains opened for ~ 1.5 ms; this is interrupted by channel closing bursts of ~ 20 $\mu$s which occur at a frequency of 1-2 closures/opening burst [@albert1993].
Channel opening is not an all or nothing event. Instead, a channel typically exhibits two conductance states, one on which it is fully opened, named a full conductance state (i.e. the active form), and one in which the channel is partially opened, named the sub-conductance state. Although the conductance rate between channels from various preparations is similar, the ratio between the two as well as their fine structure varies depending on the concentration, the agonist used and and neuronal preparation.
<!-- Immunocytochemistry using monoclonal antibodies specifie -->
<!-- In the brain, cholinergic neurons can be found in almost all parts of the brain and the optical lobe. In particular, mushroom bodies, -->
<!-- thoracic ganglia : afferent sensory neurons -->
<!-- In the peripheral primary sensory nerouns of the compound eye and the anntena. -->
<!-- Afferent sensory neurons -->
<!-- interneurones of the thoracic ganglia -->
Large quantities of achetylcholine isolated from the insect brain [@florey1963]. Further biochemical studies in which various molecules of the cholinergic synapse have been identified brain regions with the cholinergic input.
Choline acetyltransferase biosynthesis of acetylcholine, and it is thought to be a marker of cholinergic neurons.
immonocytochemistry: binding of choline acetyltransferase (ChAT) monoclonal antibodies :
<!-- Cholinergic neurons in the cortical regions of almost all regions of the insect brain -->
in the brain of locust *Locusta migratoria* [@geffard1985].
<!-- Immunocytochemistry -->
also "immunoreactivity first, in
regions of neuropile previously shown by backfilling
with cobalt to contain terminals from sensory neurones. Some roots of peripheral nerves also bind the antibody. Secondly, a small number of neurone cell bodies in the brain and thoracic ganglia are immunoreactive and we assume these belong to interneurones" and " first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the
ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies" [@lutz1987].
<!-- Biochemical techniques using monoclonal antibodies specific agains -->
immunoreactivity in almost all brain regions of *D. melanogaster* " apart from
the lobes and the peduncle of the mushroom body and
most of the first visual neuropile (lamina)" [@buchner1986].
<!-- Large quantities of achetylcholine isolated from the insect brain preparations -->
in-situ hybridization: [@barber1989] "Substantial amounts of cRNA probe for ChAT mRNA
were observed associated with cells of all cortical regions of
the optic lobe and brain. Neuropil regions of the
brain displayed insignificant amounts of hybridization with
the experimental probe and specimens incubated with the control probe did not exhibit significant
hybridization in either cortical or neuropil areas .
In peripheral nervous tissue, there was substantial labeling over primary sensory neurons in the antenna, and significant amounts of probe were associated with the
retinular cell layer of the compound eye ". the compound eye is the visual system of the fly.
<!-- also "immunoreactivity first, in -->
<!-- regions of neuropile previously shown by backfilling -->
<!-- with cobalt to contain terminals from sensory neurones. Some roots of peripheral nerves also bind the antibody. Secondly, a small number of neurone cell bodies in the brain and thoracic ganglia are immunoreactive and we assume these belong to interneurones" and " first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the -->
<!-- ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies" [@lutz1987]. -->
AND " histochemical detection of reporter gene expression " using x-gal : [@yasuyama1999].
<!-- immunoreactivity in almost all brain regions of *D. melanogaster* " apart from -->
<!-- the lobes and the peduncle of the mushroom body and -->
<!-- most of the first visual neuropile (lamina)" [@buchner1986]. -->
<!-- in-situ hybridization: [@barber1989] "Substantial amounts of cRNA probe for ChAT mRNA -->
<!-- were observed associated with cells of all cortical regions of -->
<!-- the optic lobe and brain. Neuropil regions of the -->
<!-- brain displayed insignificant amounts of hybridization with -->
<!-- the experimental probe and specimens incubated with the control probe did not exhibit significant -->
<!-- hybridization in either cortical or neuropil areas . -->
<!-- In peripheral nervous tissue, there was substantial labeling over primary sensory neurons in the antenna, and significant amounts of probe were associated with the -->
<!-- retinular cell layer of the compound eye ". the compound eye is the visual system of the fly. -->
<!-- AND " histochemical detection of reporter gene expression " using x-gal : [@yasuyama1999]. -->
" cholinergic primary sensory neurons in the Drosophila antennal system"
aceti-cholinesterase : colorimetric determination of acetylcholinesterase activity with
Histochemistry of AChE with colorimetric assay using isothiocholine:
"the optic lobes, fibers connecting the two brain hemispheres, and fiber tracts
as well as soma clusters within the protocerebrum. The calycal input regions of
the mushroom bodies were labelled, whereas the intrinsic Kenyon cells
showed no staining. Although the antennal afferents projecting into the dorsal
lobe showed strong AChE activity, projections into the antennal lobe showed
rather weak staining." [@kreissl1989] the same paper shows a-bungarotoxin (a-BTX) binding sites and AChR monoclonal antibodies with a good overlap in "d immunoreactivity
in neuropiles, tracts, somata, and the antennal nerve. The immunoreactivity
of the optic lobes coincided with the banding pattern of the AChE staining. A
particularly striking overlap of AChR immunoreactivity and AChE staining
was found in the lip neuropile of the mushroom bodies" all in the honeybee
<!-- " cholinergic primary sensory neurons in the Drosophila antennal system" -->
<!-- aceti-cholinesterase : colorimetric determination of acetylcholinesterase activity with -->
<!-- Histochemistry of AChE with colorimetric assay using isothiocholine: -->
<!-- "the optic lobes, fibers connecting the two brain hemispheres, and fiber tracts -->
<!-- as well as soma clusters within the protocerebrum. The calycal input regions of -->
<!-- the mushroom bodies were labelled, whereas the intrinsic Kenyon cells -->
<!-- showed no staining. Although the antennal afferents projecting into the dorsal -->
<!-- lobe showed strong AChE activity, projections into the antennal lobe showed -->
<!-- rather weak staining." [@kreissl1989] the same paper shows a-bungarotoxin (a-BTX) binding sites and AChR monoclonal antibodies with a good overlap in "d immunoreactivity -->
<!-- in neuropiles, tracts, somata, and the antennal nerve. The immunoreactivity -->
<!-- of the optic lobes coincided with the banding pattern of the AChE staining. A -->
<!-- particularly striking overlap of AChR immunoreactivity and AChE staining -->
<!-- was found in the lip neuropile of the mushroom bodies" all in the honeybee -->
check this paper to see evidence for cholinergic neurons "between the
sensory neurons and interneurons of the cockroach cercal sensory system " paper: " The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine"
Its action is mediated predominately by nAChRs, which are the main cholinergic receptor type in their central nervous system [@breer1987]. The presence of nAChR in various brain regions has been detected using biochemical and electrophysiological techniques on neuronal preparations extracted from the Fruit fly *Drosophila melanogaster*, honey bee *Apis mellifera* and American cocroach *Periplaneia americana*. nAChRs have been found to be expressed in the regions associated with learning, formation of memory and the sensory processing [@heisenberg1998], namely the muschroom bodies [@kreissl1989; @gu2006; @oleskevich1999]. They are also present in the insect ganglia, which connects the brain to the peripheral nervous system. In particular, they were identified in the abdominal, thoracic and the terminal ganglia [@sattelle1981; @bai1992], which are involved in the movement of wings, abdomen and legs, as well as the regulation of the anal and reproductive muscles [@smarandache-wellmann2016]. In contrast to mammals and invertebrates (Table \@ref(tab:chlinergic-nts)), insects do not express nAChRs at the neuromuscular junction.
<!-- check this paper to see evidence for cholinergic neurons "between the -->
<!-- sensory neurons and interneurons of the cockroach cercal sensory system " paper: " The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine" -->
### Biological role of nAChRs in insects
Insect nAChRs are widely expressed in the insect nervous system, where they mediate fast synaptic transmission. Their involvement in the regulation of diverse biological processes is evident from toxicity studies during which insects were exposed to nAChR agonists.
<!-- Its action is mediated predominately by nAChRs, which are the main cholinergic receptor type in their central nervous system [@breer1987]. -->
Nicotine is a naturally occurring alkaloid found in the *Solanaceae* family of plants, including tobacco [@steppuhn2004]. It is effective against plant insect pests [@david1953], and used in organic farming in form of tobacco tea [@isman2006]. Exposure of insects to lethal dose of nicotine results in symptoms characteristic for the nervous system intoxication [@chadwick1947] and include increased locomotory activity, followed by convulsions, twitching and eventual death [@carlile2006].
<!-- The presence of nAChR in various brain regions has been detected using biochemical and electrophysiological techniques on neuronal preparations extracted from the Fruit fly *Drosophila melanogaster*, honey bee *Apis mellifera* and . -->
<!-- nAChRs have been found to be expressed in the regions -->
<!-- , namely the muschroom bodies [@kreissl1989; @gu2006; @oleskevich1999]. -->
<!-- In contrast to mammals and invertebrates (Table \@ref(tab:chlinergic-nts)), insects do not express nAChRs at the neuromuscular junction. -->
<!-- ##### Electrophysiology -->
<!-- Evidence from the application of nAChR: -->
<!-- nicotine: -->
### Kinetic properties of nAChRs
Nicotinic acetylcholine receptors have three basic conformation states: the closed, the open and the disensitised state [@katz1957; @monod1965]. The question is: how does agonist binding lead to these conformational changes? Diffuculties in crystallising muscle nAChR restricted studies of receptor structure in multiple states. Whereas AChBP lack transmembrane domains therefore is not suitable for such studies.
Evidence comes from a crystal structure of structuraly related, bacterial pentameric ligand gated ion channels. These channels are not members of the cys-loop family because they lack N-termial disulphide bond. They also do not contain a large cytoplasmic loop between M3-M4 TM helices. They do however share common topology of 5 subunits, each comprising of 4 TM helices. The comparison of the crystal structure of *Erwinia* LGIC (ELGIC) and *Gloebacter* LGIC (GLGIC). They are both cation selective. GLIC is gated by protons, whereas ELIC by amines [@]
ELIC with no agonist bound - hence closed confirmation [@]
Aromatic residues in C loop form a cap above the bound agonist and by doing so they decrease the on/off rates of receptor binding [@hilf2008] this was compared to the crystal structure of the agonist-bound GLIC [@hilf2009].
Ligand binding leads to movement of the C-loop amino acids which fold over the agonist binding site, burrying it inside the protein and reducing the dissociation on/off rates of the bound agonist.
Also loop A moves towards the loop B. In complementary subunit loop F moves in, towards the agonist. This propagates further structural changes. The beta sheets rearrange. Inner beta sheet remains in the exact position, but the inner one rotates towards the centre of the pentamer.
These structural changes propagate to the level of the channel, leading to its opening.
2 possible ways in which channel opens:
Hirozontal movement of pore lining helices.
Based on the Cryo EM of the mammalian muscle nAChR in closed and open state, a hypothesis of the dynamics has been derived [@unwin1995], binding of agonist leads to rotation of 5 M2 helices. Proposed that as they move, the distance between them increases, and so the ion conductivity pathway becomes wide for the ion passage. Additionaly, this results in movement of hydrophobic residues which proclude the channel entry in the closed state of the receptor. More recently, it has been proposed that the M2 helices do not move in the unified way. Some rotate, whereas others tilt or bend [@unwin2012].
@hilf2009 - ELIC in closed state and compared to the open state - bending of felices.
More reccently, structures of representatives of Cys-loop receptors, glutamate gated chloride channel [@althoff2014] and glycine receptors [@du2015].
DESENSITISATION state is a state in which agonist affinity in higher in comparison to the resting or activated state. but ion conductance probability if lower in comparison to the resting state [@monod1965]. State in which agonist binds but there is no ion conductance [@monod1965]. Crystal structure of the human $\alpha4$/$\beta2$ receptor in desensitised state [@morales-perez2016] comparison to the structures of glycine receptors [@du2015] and GluCl channel [@althoff2014], gabaA [@miller2014] in the open state revealed difference at the interface of ECD-TM and extracellular domain loop C rotation regions of the protein and the extracellular domain which leads to occlusion of the ion channel and stabilising of the receptor in desensitising state, thereby contributing to the high affinity of the agonist to the desensitised receptor.
### Neonicotinoids target nAChRs ### {#neonicstarget}
#### Electrophysiological evidence ####{#electrophysevidence}
#### Mutations in nAChRs give rise to neonicotinoid-resistance ####{#resgenevidence}
Resistance to neonicotinoids arises from mutations in nAChR subunits. Field isolates of peach aphid *Myzus persicae* [@bass2011], the cotton aphid *Aphis gossypii* [@hirata2015; @hirata2017] and the Colorado potato beetle *Leptinotarsa decemlineata* [@szendrei2012], as well as lab-isolates of brown planthopper, *Nilaparvata lugens* [@liu2005], fruit fly *Drosophila melanogaster* [@perry2008] with decreased sensitivity to neonicotinoids have been identified. Behavioral analysis shows that their sensitivity is up to 1500-fold lower in comparison to the reference strains, as shown by the shift in LD50. Analysis of the coding genome of the resistant strains identified mutations in nAChR subunit coding sequence [@bass2011; @perry2008; @hirata2015].
#### Neonicotinoids evoke nAChR-like current in insect neuronal preparations ####{#electrophysevidence}
The effects of neonicotinoids on the neuronal transmission was investigated on insect neuronal preparations which express high levels of nAChRs.
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects. This conclusion was supported by the behavioural observation, whereby the neonicotinoid intoxication mirrors intoxication seen with cholinergic agents (Section . In response to imidacloprid, insects become hyper excited as evident by excessive pacing. They then collapse and exhibit diminishing uncoordinated leg and abdomen movement until eventual death [@sone1994; @elbart1997; @suchail2001]. Sub-lethal doses (i.e. < 4 nM) have distinct effect, such as an inhibition of feeding leading to starvation [@nauen1995; @elbart1997].
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects.
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured cocroach [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons. These data provide evidence that neonicotinoids act directly on the post-synaptic neuron in both target and non-target insects.
<!-- This phenomenon is due to the distinct conformation of nAChRs, at in which they are not capable of ion conduction [@nemecz2016]. -->
<!-- In the continual presence of agonist, the membrane potential gradually repolarises until it reaches the resting state, hovewer the next depolarisation will not occur, until the agonist are washed off and the receptor recovers from desensitisation [@katz1957]. -->
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured cocroach [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons.
Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC50 values (concentrations at which the half of the maximum current was observed) are in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(potentpests)). Neonicotinoid-induced currents were reminiscent of those induced by acetylcholine and nicotine, and were prevented by the application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine), suggesting neonicotinoid-induced currents are due to the activation of nicotinic receptors.
#### Biochemical evidence
### Mode of action of neonicotinoids ###{#moaneonicsinsects}
Neonicotinoids can have diverse mode of action. The currents produced by neonicotinoids and ACh on cultured or isolated insect neuronal preparation were compared. Neonicotinoids evoking current lower than that evoked by ACh were classed as partial agonists, those eliciting similar response were classed as true agonists, whereas those more efficacious than ACh, super-agonists. Thiacloprid and imidacloprid are partial agonists, nitenpyram, clothianidin, acetamiprid and dinotefuran are true agonists, whereas thiamethoxam has no effect on the isolated American cockroach thoracic ganglion neurons [@tan2007]. This differs from the mode of action of neonicotinoids on cultured terminal abdominal ganglion neurons of this insect. Currents produced by all neonicotinoids tested was lower than that evoked by ACh [@ihara2006], suggesting they are all partial agonists on these cells. The mode of action of neonicotinoids on the fruit fly [@brown2006] and honey bee neurons [@palmer2013] differs still, implying the presence of distinct nAChRs in different insect species and neuronal preparations.
@tomizawa1996 developed neonicotinoid agarose affinity column to isolate proteins with high binding affinity to neonicotinoids. Using Drosophila and Musca head membrane preparations, he identified three nAChR subunits as potential neonicotinoid-targets.
##### Ligand binding studies #####{#ligbinding}
#### Neonicotinoids bind with high affinity to insect nAChRs ####{#ligbinding}
<!-- @tomizawa1996 developed neonicotinoid agarose affinity column to isolate proteins with high binding affinity to neonicotinoids. Using Drosophila and Musca head membrane preparations, he identified three nAChR subunits as potential neonicotinoid-targets. -->
The binding affinity of imidacloprid to nAChRs expressed in insect membrane homogenates was assessed in the saturation ligand binding studies.
In the saturation binding experiment, various concentration of the labelled ligand is added to the preparation and the concentration of the ligand at the equilibrium is determined. This is then used to derive the binding constant (as a measure of dissociation constant, Kd). Here, it was used to define the binding strength of neonicoinoids to insect nAChRs (Table \@ref(tab:bindignrecombinant)).
<!-- In the saturation binding experiment, various concentration of the labelled ligand is added to the preparation and the concentration of the ligand at the equilibrium is determined. This is then used to derive the binding constant (as a measure of dissociation constant, Kd). Here, it was used to define the binding strength of neonicoinoids to insect nAChRs (Table \@ref(tab:bindignrecombinant)). -->
Imidacloprid and thiamethoxam bind with high affinity to proteins in the whole membrane preparations of the domestic fly and aphids with the Kd in the low nM range [@liu1993; @wellmann2004; @liu2005]. Interestingly, two binding affinities have been derived from the imidacloprid study in the brown planthopper and pea aphid [@wellmann2004; @taillebois2014] suggesting there are two binding sites in these animals.
......@@ -441,17 +575,78 @@ library(kableExtra)
threeparttable = T)
```
In addition to the saturation studies, the competitive ligand binding studies were carried out. In the the competitive ligand binding studies, biological preparation is incubated with radiolabelled ligand. The ability of various concentrations of unlabeled ligand is measured to define its equilibrium inhibition constant (Ki). This method informs both on the affinity and on the interactions between ligands.
In addition to the saturation studies, the competitive ligand binding studies were carried out.
<!-- In the the competitive ligand binding studies, biological preparation is incubated with radiolabelled ligand. The ability of various concentrations of unlabeled ligand is measured to define its equilibrium inhibition constant (Ki). This method informs both on the affinity and on the interactions between ligands. -->
Various concentrations of neonicotinoid prototype isothiaocynate were incubated with the homogenate of fruit fly *Drosophila melanogaster* and a homogenate of the abdominal nerve cords of *Periplaneta americana* before the exposure to radiolabelled nAChR antagonist $\alpha$-bgtx [@gepner1978]. Isothiaocynate inhibited binding of $\alpha$-bgtx in the concentration dependent manner [@gepner1978], suggesting the two compounds share the binding site. Similarly, imidacloprid has been shown to displace $\alpha$-bgtx from brain membrane preparations from honey bee *Apis mellifera* [@tomizawa1992; @tomizawa1993], *Drosophila melanogaster* [@zhang2004], house fly *Musca domestica* and isolated cockroach nerve cords [@bai1991].
#### Genetic evidence ####{#resgenevidence}
#### High affinity of neonicotinoids to heterologously expressed insect-chimera receptors ####{#chimerareceptors}
Resistance to neonicotinoids arises from mutations in nAChR subunits. Field isolates of peach aphid *Myzus persicae* [@bass2011], the cotton aphid *Aphis gossypii* [@hirata2015; @hirata2017] and the Colorado potato beetle *Leptinotarsa decemlineata* [@szendrei2012], as well as lab-isolates of brown planthopper, *Nilaparvata lugens* [@liu2005], fruit fly *Drosophila melanogaster* [@perry2008] with decreased sensitivity to neonicotinoids have been identified. Behavioral analysis shows that their sensitivity is up to 1500-fold lower in comparison to the reference strains, as shown by the shift in LD50. Analysis of the coding genome of the resistant strains identified mutations in nAChR subunit coding sequence [@bass2011; @perry2008; @hirata2015].
### Mode of action of neonicotinoids ###{#moaneonicsinsects}
Due to the difficulties in the expression of native insect receptors (Section \@ref(expressionfail)), the binding affinity of neonicotinoids was determined in hybrid receptors, consisting of insect $\alpha$-subunit and vertebrate $\beta$ subunit. $\beta2$ from rat and chicken has been shown to enable recombinant expression of several insect $\alpha$ subunits in cell lines.
Neonicotinoids can have diverse mode of action. The currents produced by neonicotinoids and ACh on cultured or isolated insect neuronal preparation were compared. Neonicotinoids evoking current lower than that evoked by ACh were classed as partial agonists, those eliciting similar response were classed as true agonists, whereas those more efficacious than ACh, super-agonists. Thiacloprid and imidacloprid are partial agonists, nitenpyram, clothianidin, acetamiprid and dinotefuran are true agonists, whereas thiamethoxam has no effect on the isolated American cockroach thoracic ganglion neurons [@tan2007]. This differs from the mode of action of neonicotinoids on cultured terminal abdominal ganglion neurons of this insect. Currents produced by all neonicotinoids tested was lower than that evoked by ACh [@ihara2006], suggesting they are all partial agonists on these cells. The mode of action of neonicotinoids on the fruit fly [@brown2006] and honey bee neurons [@palmer2013] differs still, implying the presence of distinct nAChRs in different insect species and neuronal preparations.
Mammalian $\alpha4$/$\beta2$ receptor expresses well in Xenopus oocytes [@cooper1991] and cell lines [@lansdell2000] has low affinity to imidacloprid (Kd >1000 $\mu$M) [@lansdell2000]. Chimera of rat $\beta2$ and $\alpha$ subunits from the fruit fly *Drosophila melanogaster* [@lansdell2000], aphid *Myzus Persicae* [@huang1999], planthopper *Nilaparvata lugens* [@liu2009], cat flea *Ctenocephalides felis* [@bass2006] and sheep blowfly *Lucilia cuprina* [@dederer2011] have been generated. It needs to be noted that the potency of neonicotinoids on these receptors is unknown due to the lack of reported data, suggesting these receptors are not functional. However, their pharmacological profiles have been determined using saturation ligand binding studies [@hulme2010] (Table \@ref(tab:bindignrecombinant)).
The affinity of neonicotinoids to insect-chimera rectors varies, depending on the identity of the $\alpha$ subunit. Imidacloprid did not bind to Mp$\alpha1$/rat$\beta2$ receptor, whereas its Ki at Mp$\alpha2$ and Mp$\alpha3$-containing receptor was 3 and 2.8 nM, respectively [@huang1999]. Four to five fold-difference between the most and least susceptible *Drosophila melanogaster* and *Ctenocephalides felis* receptor assemblies were also identified [@lansdell2000; @bass2006]
Imidacloprid exhibits the highest affinity against target pest *Myzus Persicae* with the lowest reported Ki of 2.8 nM on $\alpha3$/$\beta2$ receptor [@huang1999]. It binds less tightly to the non-target insect, the fruit fly nAChRs; the Kd values range from 8.4 to 34.9 nM [@lansdell2000].
```{r bindignrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
footnotez <- ("Receptors were expressed in insect S2 cell line")
footnotey <- ("Rn = Rattus norvegicus (rat), Dm = *Drosophila melanogaster (fruit fly), Mp = Myzus persicae (aphid), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), N/B = no binding,")
bindingrecombinant <- data.frame(
Receptor = c("Rn$\\alpha4\\beta2$", "Dm$\\alpha1$/Rn$\\beta2$", "Dm$\\alpha2$/Rn$\\beta2$", "Dm$\\alpha3$/Rn$\\beta2$", "Mp$\\alpha1$/Rn$\\beta2$", "Mp$\\alpha2$/Rn$\\beta2$", "Mp$\\alpha3$/Rn$\\beta2$", "Mp$\\alpha4$/Rn$\\beta2$", "Nl$\\alpha1$/Rn$\\beta2$", "Cf$\\alpha1$/Dm$\\alpha2$/Rn$\\beta2$", "Cf$\\alpha3$/Dm$\\alpha2$/Rn$\\beta2$"),
Kd= c(">1000", "34.9", "20", "8.4", "N/B", "3", "2.8", "N/B", "24.3", "141", "28.7"),
References = c("Lansdell and Millar, 2000", "", "", "", "Huang et al., 1999", "", "", "", "Liu et al., 2005", "Bass et al. 2006", ""))
bindingrecombinant %>%
mutate_all(linebreak) %>%
kable("latex", align = "c", booktabs = TRUE, escape = F,
col.names = linebreak(c("Receptor", "Kd\n(nM)", "Rerefence")),
caption = 'Binding affinity of imidacloprid to recombinant insect-hybrid receptors') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote(notation = "none", c(footnotez, footnotey),
threeparttable = T)
```
#### High potency of neonicotinoids on heterologously expressed insect-mammalian hybrid receptors
The potency of neonicotinoids on insect-mammal hybrid nAChRs have been determined using electrophysiological techniques. Due to the difficulties in heterologous expression of native insect receptors (Section \@ref), $\alpha$ nAChR subunit from the fruit fly, the cat flea and the planthopper were co-expressed with rat or mouse $\beta2$. The responses of the formed channel were recorded in the presence of various neonicotinoids: cyanoamidines clothianidin and imidacloprid, nitroguanidines thiacloprid and acetamiprid and nitromethylene nitenpyram.
Depolarising current was recorded from cells expressing insect-hybrid nAchRs in responses to all tested neonicotinoids. This response was dose-dependent. The potency of neonicotinoids varied, as indicated by the EC50 value, between 0.04 and 45.8 $\mu$M, however it is generally in the region of 1 $\mu$M.
The rank order of potency of cyanoamidines, nitroguanidine and nitromethylene differs, depending on the receptor identity. For example, in imidacloprid and clothianidin are the most potent on the fruit fly $\alpha1$ containing receptors [@dederer2011], whereas planthopper $\alpha3\alpha8$ hybrid, thiacloprid is the most efficacious [@yixi2009]. Nitenpyram has consistently the highest EC50.
```{r potencyrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
footnotew <- ("Receptors were expressed in Xenopus oocytes")
footnotex <- ("Rn = Rattus norvegicus (rat), Gg = Gallus gallus (chicken), Dm = *Drosophila melanogaster (fruit fly), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), Lc = Lucilia cuprina (sheep blowfly)")
potencyrecombinant <- data.frame(
Receptor = c("Nl$\\alpha1$/Rn$\\beta2$", "Nl$\\alpha2$/Rn$\\beta2$", "Nl$\\alpha3$/Rn$\\beta2$", "Nl$\\alpha3\\alpha8$/Rn$\\beta2$", "", "", "", "Dm$\\alpha1$/Gg$\\beta2$", "", "", "", "Dm$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha1$/Gg$\\beta2$", "", "", "", "Cf$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha4$/Gg$\\beta2$", "", "", ""),
Compound = c("Imidacloprid", "Imidacloprid", "Imidaclorprid", "Imidacloprid", "Clothianidin", "Thiacloprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram"),
EC50 = c("61", "870", "350", "3.2", "5.1", "2.8", "5.6", "0.04", "0.34", "0.23", "0.4", "0.84", "5.4", "2", "35.4", "0.02", "0.15", "0.11", "0.63", "1.31", "1.65", "2.63", "24.4", "13.8", "21.3", "9.4", "45.8"),
References = c("Liu et al. 2009", "", "", "Yixi et al. 2009", "", "", "", "Dederer et al. 2011", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", ""))
potencyrecombinant %>%
mutate_all(linebreak) %>%
kable("latex", align = "c", booktabs = TRUE, escape = F,
col.names = linebreak(c("Receptor", "Compound", "EC50\n($\\mu$M)", "Rerefence")),
caption = 'The potency of neonicotinoids on recombinantly expressed insect hybrid nAChRs.') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote(notation = "none", c(footnotew, footnotex),
threeparttable = T)
```
<!-- Based on the expression of several potential assemblies have been identified. -->
<!-- Subunits with various degree of sensitivity to neonicotinoids, suggesting some and not all receptors confer neonicotinoid sensitivity. -->
<!-- cultured Drosophila CNS cholinergic neurons, whereas imidacloprid is a partial agonist there . -->
......@@ -504,100 +699,31 @@ library(kableExtra)
nAChR are assemblies of 5 different or identical receptor subunits (Section \@ref(structure)). Each subunit is encoded by a separate gene and is classified as either $\alpha$ or non-$\alpha$, depending on the primary amino acid sequence, whereby $\alpha$ subunits contain a disulphide bond formed between the adjacent cysteines in the ligand binding domain (Figure \@ref(fig:structure-nachr-label)). Genome sequencing projects enabled identification of nAChR subunit families in several insect species. Fruit fly and model organism *Drosophila melanogaster* has 10 subunits, 7 of which are $\alpha$ ($\alpha1-7$) and 3 are $\beta$ ($\beta1-3$) [@adams2000a; @sattelle2005]. There are 11 subunits in the beneficial insect honeybee *A. mellifera* ($\alpha1-9$, $\beta1-2$) [@jones2006a; @consortium2006], 12 subunits in the pest red flour beetle *Tribolium castaneum* ($\alpha1-11$,, $\beta1$) [@consortium2008] and 7 in the Pea Aphid, *Acyrthosiphon pisum* ($\alpha1-6$, $\beta1-2$) [@yi-peng2013; @Consortium2010]. With the aid of molecular cloning techniques, equivalent subunits have been identified in many other insects, including cat flea *Ctenocephalides felis* [@bass2006] and green peach aphid *Myzus persicae* [@huang2000]. Amino acid sequence alignment of equivalent subunits revealed that they are highly conserved, with sequence identity typically greater than 60 % [@jones2010].
Insect nAChR gene families are among the least diverse when compared to other animal phyla. Mammals express 17 subunits: $\alpha1-10$, $\beta1-4$, $\delta$, $\gamma$ and $\epsilon$ [@millar2009] and there are 29 subunits in the representative of the phylum *Nematoda, C. elegans* [@jones2007b].
Insect nAChR gene families are among the least diverse when compared to other animal phyla. Mammals express 17 subunits: $\alpha1-10$, $\beta1-4$, $\delta$, $\gamma$ and $\epsilon$ [@millar2009] and there are 29 subunits in the representative of the phylum *Nematoda, C. elegans* [@jones2007a].
### RIC-3 improves recombinant nAChR assembly ###{#ric3insect}
### Diffuculties in heterologous expression of insect nAChRs
<!-- There are different receptor types in insects. -->
<!-- https://radar.brookes.ac.uk/radar/file/c59cbdb5-d171-49e0-b0e4-101c261c72ed/1/fulltext.pdf -->
To identify which subunits assemble to form functional receptors, recombinant expression techniques were used.
Recombinant expression is a technique receptor stoichiometry and function can be studied in a heterologous system. cDNA is injected into the Xenopus oocytes, or used to transfect insect or mammalian cell line. Using internal cellular machinery, it is transcribed, translated and processed to the surface of the cell. Should a protein form, cell-surface expression can be detected using biochemical approaches (such as ligand binding studies), whereas function studied by means of electrical recordings. These approaches were utilised to identify the major receptor assemblies in mammals, nematode and fish (Table \@ref(tab:chlinergic-nts)).
To identify which subunits assemble to form functional receptors, recombinant expression techniques were used. Recombinant expression is a technique receptor stoichiometry and function can be studied in a heterologous system. cDNA is injected into the Xenopus oocytes, or used to transfect insect or mammalian cell line. Using internal cellular machinery, it is transcribed, translated and processed to the surface of the cell. Should a protein form, cell-surface expression can be detected using biochemical approaches (such as ligand binding studies), whereas function studied by means of electrical recordings. These approaches were utilised to identify the major receptor assemblies in mammals, nematode and fish (Table \@ref(tab:chlinergic-nts)).
<!-- To determine which insect nAChR subunits come together to form a functional receptor, various nAChR subunits were injected into the expression systems (*Xenopus oocytes* or cell lines) to determine whether cell surface expression and receptor function can be obtained. -->
To determine which insect subunits form functional nAChRs, @lansdell2012 transfected cultured insect cells with over 70 *Drosophila melanogaster* nAChR subunit cDNAs either singularly or in combinations. No cell surface was achieved, as shown by the radiolabelled ligand binding studies. Difficulties in expression of *Drosophila melanogaster* were also encountered in Xenopus oocytes [@lansdell2012] and mammalian cell lines [@lansdell1997]. The attempts to express receptors from other species were also largely unsuccessful. No ligand binding and/or agonist evoked currents were detected from cells transfected with genes encoding for the brown planthopper *Nilaparvata lugens* [@liu2005; @liu2009; @yixi2009], cat flea *Ctenocephalides felis* [@bass2006], aphid *Myzus persicae* nAChR subunits [@huang2000] and brown dog tick *Rhipicephalus sanguineus* [@lees2014]. Homomeric Locust *Schistocerca gregaria* $\alpha1$ [@marshall1990], *Myzus Persicae* $\alpha1$ and *Myzus Persicae* $\alpha2$ [@sgard1998] produced receptors with nAChR-like pharmacological and electrophysiological characteristics, however the channel-generated currents were of low amplitude, and the expression inconsistent.
Difficulties in recombinant receptor expression highlight the complexity of receptor formation (Figure \@ref(fig:turnover-label)). Assembly and oligomerisation are critical steps in the receptor maturation. These steps require a number of ER and Golgi resident chaperons. RIC-3 (resistant to inhibitors of cholinesterase-3) is an evolutionary conserved, ER-residing [@roncarati2006; @alexander2010] transmembrane protein [@wang2009]. The role of RIC-3 in receptor expression was first described using *C. elegans* and human receptors (Section \@ref(ric-3nacho) and \@ref(ric-3celegans)). More recently, its role in receptor folding and maturation of insect nAChRs has been demonstrated. Co-expression of Dm$\alpha2$-containing and Dm$\alpha5$/$\alpha7$ receptors with RIC-3 improved [@lansdell2008], and in some instances enabled expression in otherwise non-permissible systems [@lansdell2012]. Up to 3.5-fold increase in specific binding of radiolabelled antagonist was noted in insect cells co-transfected with RIC-3, suggesting the presence of greater number of folded receptors on the cell surface [@lansdell2008]. Expressed receptors have been also shown to be functional. In Xenopus oocytes, ionic currents were detected in response to acetylcholine in cells co-expressing RIC-3 [@lansdell2012].
RIC-3 enabled identification of potential nAChR in insects: homomeric $\alpha5$ and $\alpha7$ and heteromeric $\alpha5$/$\alpha7$. The identity of other insect receptors is unknown.
(ref:turnover) **Nicotinic acetylcholine receptor turnover.** Synthesised nAChR subunit peptides undergo folding and oligomerisation in the ER. Correctly folded receptors are transported into the Golgi (1). Misfolded subunits and misassembled receptors are retained in the ER and eventually degraded (2). Receptors transported to the Golgi undergo maturation to be shipped to the plasma membrane (4). Receptors in the plasma membrane are eventually degraded or recycled. Receptors are first packed into the endosome (4) and transported to the lysosome or proteosome for degradation (5) or re-inserted into the plasma membrane (6).
```{r turnover-label, fig.cap="(ref:turnover)", fig.scap= 'Nicotinic acetylcholine receptor turnover.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/nAChR_turnover.png")
```
### Insect sensitivity to neonicotinoids in recombinant systems
Difficulties in expression of recombinant insect nAChRs (Section \@ref(expressionfail)) hiders their pharmacological analysis and identification of receptors sensitive to neonicotinoids. Insect-mammal hybrid receptors served as a platform to investigate the potency and affinity of neonicotinoids.
#### High affinity of neonicotinoids to insect-chimera receptors ####{#chimerareceptors}
Mammalian $\alpha4$/$\beta2$ receptor expresses well in Xenopus oocytes [@cooper1991] and cell lines [@lansdell2000] and it has low affinity to imidacloprid (Kd >1000 $\mu$M) [@lansdell2000]. $\beta2$ from rat and chicken has been shown to enable recombinant expression of several insect $\alpha$ subunits in cell lines. Chimera of rat $\beta2$ and $\alpha$ subunits from the fruit fly *Drosophila melanogaster* [@lansdell2000], aphid *Myzus Persicae* [@huang1999], planthopper *Nilaparvata lugens* [@liu2009], cat flea *Ctenocephalides felis* [@bass2006] and sheep blowfly *Lucilia cuprina* [@dederer2011] have been generated. It needs to be noted that the potency of neonicotinoids on these receptors is unknown due to the lack of reported data, suggesting these receptors are not functional. However, their pharmacological profiles have been determined using saturation ligand binding studies [@hulme2010] (Table \@ref(tab:bindignrecombinant)).
The affinity of neonicotinoids to insect-chimera rectors varies, depending on the identity of the $\alpha$ subunit. Imidacloprid did not bind to Mp$\alpha1$/rat$\beta2$ receptor, whereas its Ki at Mp$\alpha2$ and Mp$\alpha3$-containing receptor was 3 and 2.8 nM, respectively [@huang1999]. Four to five fold-difference between the most and least susceptible *Drosophila melanogaster* and *Ctenocephalides felis* receptor assemblies were also identified [@lansdell2000; @bass2006]
Imidacloprid exhibits the highest affinity against target pest *Myzus Persicae* with the lowest reported Ki of 2.8 nM on $\alpha3$/$\beta2$ receptor [@huang1999]. It binds less tightly to the non-target insect, the fruit fly nAChRs; the Kd values range from 8.4 to 34.9 nM [@lansdell2000], suggesting it is selectively toxic to pest insects.
```{r bindignrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
footnotez <- ("Receptors were expressed in insect S2 cell line")
footnotey <- ("Rn = Rattus norvegicus (rat), Dm = *Drosophila melanogaster (fruit fly), Mp = Myzus persicae (aphid), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), N/B = no binding,")
bindingrecombinant <- data.frame(
Receptor = c("Rn$\\alpha4\\beta2$", "Dm$\\alpha1$/Rn$\\beta2$", "Dm$\\alpha2$/Rn$\\beta2$", "Dm$\\alpha3$/Rn$\\beta2$", "Mp$\\alpha1$/Rn$\\beta2$", "Mp$\\alpha2$/Rn$\\beta2$", "Mp$\\alpha3$/Rn$\\beta2$", "Mp$\\alpha4$/Rn$\\beta2$", "Nl$\\alpha1$/Rn$\\beta2$", "Cf$\\alpha1$/Dm$\\alpha2$/Rn$\\beta2$", "Cf$\\alpha3$/Dm$\\alpha2$/Rn$\\beta2$"),
Kd= c(">1000", "34.9", "20", "8.4", "N/B", "3", "2.8", "N/B", "24.3", "141", "28.7"),
References = c("Lansdell and Millar, 2000", "", "", "", "Huang et al., 1999", "", "", "", "Liu et al., 2005", "Bass et al. 2006", ""))
bindingrecombinant %>%
mutate_all(linebreak) %>%
kable("latex", align = "c", booktabs = TRUE, escape = F,
col.names = linebreak(c("Receptor", "Kd\n(nM)", "Rerefence")),
caption = 'Binding affinity of imidacloprid to recombinant insect-hybrid receptors') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote(notation = "none", c(footnotez, footnotey),
threeparttable = T)
```
To determine which insect subunits form functional nAChRs, @lansdell2012 transfected cultured insect cells with over 70 *Drosophila melanogaster* nAChR subunit cDNAs either singularly or in combinations. No cell surface was achieved, as shown by the radiolabelled ligand binding studies. Difficulties in expression of *Drosophila melanogaster* were also encountered in Xenopus oocytes [@lansdell2012] and mammalian cell lines [@lansdell1997]. The attempts to express receptors from other species were also largely unsuccessful. No ligand binding and/or agonist evoked currents were detected from cells transfected with genes encoding for the nAChR subunits of brown planthopper *Nilaparvata lugens* [@liu2005; @liu2009; @yixi2009], cat flea *Ctenocephalides felis* [@bass2006], aphid *Myzus persicae* [@huang2000] and brown dog tick *Rhipicephalus sanguineus* [@lees2014]. Homomeric Locust *Schistocerca gregaria* $\alpha1$ [@marshall1990], *Myzus Persicae* $\alpha1$ and *Myzus Persicae* $\alpha2$ [@sgard1998] produced receptors with nAChR-like pharmacological and electrophysiological characteristics, however the channel-generated currents were of low amplitude, and the expression was inconsistent.
#### High potency of neonicotinoids on insect-hybrid receptors
##### Importance of chaperon proteins in heterologous expression of nAChRs ###{#ric3insect}
The potency of neonicotinoids on insect-mammal hybrid nAChRs have been determined using electrophysiological techniques. $\alpha$ nAChR subunit from the fruit fly, the cat flea and the planthopper were co-expressed with rat or mouse $\beta2$. The responses of the formed channel were recorded in the presence of various neonicotinoids: cyanoamidines clothianidin and imidacloprid, nitroguanidines thiacloprid and acetamiprid and nitromethylene nitenpyram.
Difficulties in recombinant receptor expression highlight the complexity of receptor formation. Assembly and oligomerisation are critical steps in the receptor maturation [@brodsky1999]. Co-expression of mammalian and *C. elegans* nAChRs with chaperon proteins highlight the critical role of ER and Golgi resident chaperons in receptor maturation. @boulin2008 demonstrated that three chaperon proteins are necessary for the expression of *C. elegans* muscle-type receptors in Xenopus oocytes: *C. elegans* UNC-50, UNC-74 and RIC-3 (more details on these proteins can be found in Sections \@ref(ric-3celegans), \@(unc-50) and \@ref(unc74)); ligand binding and agonist-evoked currents were abolished upon exclusion of any of the three proteins. RIC-3 also improves the cell surface expression of the second type of the BWM *C. elegans* receptor [@ballivet1996] and in the of neuron-type receptor in Xenopus oocytes [@halevi2002]. It also plays a role in the maturation of human receptor in Xenopus oocytes and cell lines (Section \@ref(ric-3nacho). More recently, RIC-3 has been shown to influence folding and maturation of insect nAChRs. Co-expression of Dm$\alpha2$-containing and Dm$\alpha5$/$\alpha7$ receptors with RIC-3 improved [@lansdell2008], and in some instances enabled expression in otherwise non-permissible systems [@lansdell2012]. Up to 3.5-fold increase in specific binding of radiolabelled antagonist was noted in insect cells co-transfected with RIC-3, suggesting the presence of greater number of folded receptors on the cell surface [@lansdell2008]. Expressed receptors have been also shown to be functional. In Xenopus oocytes, ionic currents were detected in response to acetylcholine in cells co-expressing RIC-3 [@lansdell2012].
Upward (depolarising) current was recorded from cells expressing insect-hybrid nAchRs in responses to all tested neonicotinoids. This response was dose-dependent. The potency of neonicotinoids varied, as indicated by the EC50 value, between 0.04 and 45.8 $\mu$M, however it is generally in the region of 1 $\mu$M.
<!-- <!-- RIC-3 enabled identification of potential insect nAChRs. Co expression of Drosophila $\alpha5$ and $\alpha7$ either singlularly or in combonation, gave rise to cell-surface expression, as shown by radiolabelled ligand binding [@lansdell] and heteromeric $\alpha5$/$\alpha7$. The identity of other insect receptors is unknown. -->
The rank order of potency of cyanoamidines, nitroguanidine and nitromethylene differs, depending on the receptor identity. For example, in imidacloprid and clothianidin are the most potent on the fruit fly $\alpha1$ containing receptors [@dederer2011], whereas planthopper $\alpha3\alpha8$ hybrid, thiacloprid is the most efficacious [@yixi2009]. Nitenpyram has consistently the highest EC50.
<!-- (ref:turnover) **Nicotinic acetylcholine receptor turnover.** Synthesised nAChR subunit peptides undergo folding and oligomerisation in the ER. Correctly folded receptors are transported into the Golgi (1). Misfolded subunits and misassembled receptors are retained in the ER and eventually degraded (2). Receptors transported to the Golgi undergo maturation to be shipped to the plasma membrane (4). Receptors in the plasma membrane are eventually degraded or recycled. Receptors are first packed into the endosome (4) and transported to the lysosome or proteosome for degradation (5) or re-inserted into the plasma membrane (6). -->
```{r potencyrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
<!-- ```{r turnover-label, fig.cap="(ref:turnover)", fig.scap= 'Nicotinic acetylcholine receptor turnover.', fig.align='center', echo=FALSE} -->
footnotew <- ("Receptors were expressed in Xenopus oocytes")
footnotex <- ("Rn = Rattus norvegicus (rat), Gg = Gallus gallus (chicken), Dm = *Drosophila melanogaster (fruit fly), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), Lc = Lucilia cuprina (sheep blowfly)")
potencyrecombinant <- data.frame(
Receptor = c("Nl$\\alpha1$/Rn$\\beta2$", "Nl$\\alpha2$/Rn$\\beta2$", "Nl$\\alpha3$/Rn$\\beta2$", "Nl$\\alpha3\\alpha8$/Rn$\\beta2$", "", "", "", "Dm$\\alpha1$/Gg$\\beta2$", "", "", "", "Dm$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha1$/Gg$\\beta2$", "", "", "", "Cf$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha4$/Gg$\\beta2$", "", "", ""),
Compound = c("Imidacloprid", "Imidacloprid", "Imidaclorprid", "Imidacloprid", "Clothianidin", "Thiacloprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram"),
EC50 = c("61", "870", "350", "3.2", "5.1", "2.8", "5.6", "0.04", "0.34", "0.23", "0.4", "0.84", "5.4", "2", "35.4", "0.02", "0.15", "0.11", "0.63", "1.31", "1.65", "2.63", "24.4", "13.8", "21.3", "9.4", "45.8"),
References = c("Liu et al. 2009", "", "", "Yixi et al. 2009", "", "", "", "Dederer et al. 2011", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", ""))
potencyrecombinant %>%
mutate_all(linebreak) %>%
kable("latex", align = "c", booktabs = TRUE, escape = F,
col.names = linebreak(c("Receptor", "Compound", "EC50\n($\\mu$M)", "Rerefence")),
caption = 'The potency of neonicotinoids on recombinantly expressed insect hybrid nAChRs.') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote(notation = "none", c(footnotew, footnotex),
threeparttable = T)
```
Based on the expression of several potential assemblies have been identified.
Subunits with various degree of sensitivity to neonicotinoids, suggesting some and not all receptors confer neonicotinoid sensitivity.
<!-- knitr::include_graphics("fig/general_intro/png/nAChR_turnover.png") -->
<!-- ``` -->
<!-- clothiandidin on the abdominal ganglion [@thany2009] and imidacloprid on the Colorado potato beetle (CPB), . -->
......@@ -630,15 +756,16 @@ Subunits with various degree of sensitivity to neonicotinoids, suggesting some a
<!-- "connections between afferents sensory neurons with interneurons or with motoneurons in several insects such as the cockroach" -->
### Recombinant receptors
<!-- ### Recombinant receptors -->
Recombinant insect nAChR are notoriously difficult to express. Several interventions have been tested including expression of hybrid receptor in which insect subunits have been co-expressed with mammalian ones. It need to be noted that this method has several limitations. First, hybrid receptors are not biologically relevant, thus conclusions from these studies should be drawn with caution. Second, some some of the expressed receptors may be folded, but not functional. Lastly, this method enabled expression of only a handful of receptors, thus most remained uncharacterised. This hinders their pharmacological characterisation and identification of subunits important in conferring the agricultural role of neonicotinoids. Heterologous expression of nAChR from insects and other species would allow for the characterisation of the interactions of these proteins with neonicotinoids to better define their mode of action and selective toxicity. Development of the platform in which the heterologous expression of insect nAChRs could be achieved, would open the door to screening of novel insecticides, to combat emerging and spreading neonicotinoid-resistance (Section \@ref(resgenevidence)) and @charaabi2018). In addition, by expressing nAChRs from pest and other species identification of compounds with no adverse effects on beneficial insects and other biologically important species may be achieved. Model organism *C. elegans* is a system in which the mode of action and the selective toxicity can be studied.
<!-- Recombinant insect nAChR are notoriously difficult to express. Several interventions have been tested including expression of hybrid receptor in which insect subunits have been co-expressed with mammalian ones. It need to be noted that this method has several limitations. First, hybrid receptors are not biologically relevant, thus conclusions from these studies should be drawn with caution. Second, some some of the expressed receptors may be folded, but not functional. Lastly, this method enabled expression of only a handful of receptors, thus most remained uncharacterised. This hinders their pharmacological characterisation and identification of subunits important in conferring the agricultural role of neonicotinoids. Heterologous expression of nAChR from insects and other species would allow for the characterisation of the interactions of these proteins with neonicotinoids to better define their mode of action and selective toxicity. Development of the platform in which the heterologous expression of insect nAChRs could be achieved, would open the door to screening of novel insecticides, to combat emerging and spreading neonicotinoid-resistance (Section \@ref(resgenevidence)) and @charaabi2018). In addition, by expressing nAChRs from pest and other species identification of compounds with no adverse effects on beneficial insects and other biologically important species may be achieved. Model organism *C. elegans* is a system in which the mode of action and the selective toxicity can be studied. -->
## Overview of *C. elegans*
## *C. elegans* a model platform for expression of nAChRs
As indicated, the expression of insect receptors is limited due to diffuculties in expression in and cellular environment required . have been studied in model organism
*C. elegans* is a transparent non-parasitic nematode, inhabiting temperate soil environments. This worm was first described as a new species in 1900 [@maupas1900] and named *Caenorhabditis elegans* Greek *caeno* meaning recent, *rhabditis* meaning rod-like and Latin *elegans* meaning elegant. The natural isolate of this species was extracted from the compost heap in Bristol by Sydney Brenner in 1960's and named N2. Since, *C. elegans* has become a valued lab tool and a model organism due to ethical, economical and biological reasons. In contrast to vertebral organisms, *C. elegans* is not protected under most animal research legislation. The cost of use is low, due to the cost of purchase (~$6/strain), maintenance, fast life cycle and high fertility of these animals. *C. elegans* has also is also the first multicellular organism to have the whole genome sequenced [@consortium1998] and the neuronal network has been mapped [@white1986]. It has an advantage over other model organisms in that its nervous system is relatively simple and it is amenable to genetic manipulations.
## General biology ##{#genbiology}
### General biology of *C. elegans* ##{#genbiology}
*C. elegans* exists as a male and hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9 % of worms are hermaphrodites, which self-fertilize their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15^o^C, it takes 5.5 days from egg-fertilization to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25 deg;C, respectively (Figure \@ref(fig:life-cycle-label)). At 20 degrees, hermaphrodite lay eggs 2.5 hours after the fertilisation. 8 hours later the embryo hatches as a larvae in the first stage of its development (L1). In the presence of food, larvae develops into an adult through three further developmental stages, namely L2, L3 and L4. The transition between each larval stage is marked by a process of maulting, during which the old cuticle is shed and replaced by a new one. In the absence of food, developing L2 and L3 worms enter the dauer stage. The worms can remain arrested at this low metabolic activity state for up to several weeks, and will develop into adults, should the food re-appear. Hermaphrodites remain fertile for the first three days of their adulthood. Their eggs can be fertilised internally with the sperm produced by the hermaphrodite, or, if there are males available, by mating. Unmated worm can lay up to 350 eggs, whereas mated over a 1000 eggs. Figure \@ref(fig:life-cycle-label) illustrated the full *C. elegans* life cycle.
......@@ -674,19 +801,13 @@ EPG (electropharyngeogram) is an extracellular electrical recording from the pha
### Mode of action studies
*C. elegans* is amenable to genetic manipulations. There is a range of genetic techiques available to generate mutant strains, in which the expression of a certain protein is greatly reduced or eliminated [@boulin2012]. Using these techniques, hundreds of mutant strains have been generated. These strains have been deposited and are available for purchase from the Caenorhabditis Genetics Center (CGC). Behaviour analysis of mutant strains allows for the identification of proteins important in the regulation of many aspects of worm behaviour; an approach used for the mode of action studies.
*C. elegans* is amenable to genetic manipulations. There is a range of genetic techiques available to generate mutant strains, in which the expression of a certain protein is greatly reduced or eliminated [@boulin2012]. Using these techniques, hundreds of mutant strains have been generated. These strains have been deposited and are available from the Caenorhabditis Genetics Center (CGC). Behaviour analysis of mutant strains allows for the identification of proteins important in the regulation of many aspects of worm behaviour; an approach used for the mode of action studies.
## Cholinergic neurotransmission regulates feeding, locomotion and reproduction in *C. elegans* ## {#cholinergicneurotransmissioninworms}
### Acetylcholine regulates feeding, locomotion and reproduction in *C. elegans* ## {#cholinergicneurotransmissioninworms}
Many of the *C. elegans* behaviours are regulated by acetylcholine, which is the major neurotransmitter in the nervous system of *C. elegans*. This is evident for the behavioural analysis of mutant strains in which acetylcholine neurotransmission is affected, as well as from the pharmacological effects of cholinergic agents on the behaviour of worms.
### Evidence from behavioural analysis
The synthesis and packing of acetylcholine into synaptic vesicles are essential steps in cholinergic neurotransmission. These functions are mediated by several proteins (Figure \@ref(fig:cholsynapsecelegand-label)). Choline acetyltransferase (ChAT) encoded by the cha-1 gene catalyses the formation of acetylcholine [@rand1985]. Vesicular acetylcholine transferase (VAChT) encoded by unc-17 loads acetylcholine into synaptic vesicles [@alfonso1993]. Null mutations of these genes are lethal due to the inhibition of worm's locomotion and feeding and its eventual death due to starvation [@rand1989; @alfonso1993]. Polymorphic ChAT and VAChT mutants in which the expression is reduced, but not abolished, revealed somewhat opposite phenotype. The pharyngeal pumping both in the presence and absence of food was reduced [@dalliere2015] the movement highly uncoordinated and jerky [@rand1984], whereas egg-laying increased [@bany2003].
### Pharmacological evidence
#### Aldicarb
The synthesis and packing of acetylcholine into synaptic vesicles are essential steps in cholinergic neurotransmission. These functions are mediated by several proteins. Choline acetyltransferase (ChAT) encoded by the cha-1 gene catalyses the formation of acetylcholine [@rand1985]. Vesicular acetylcholine transferase (VAChT) encoded by unc-17 loads acetylcholine into synaptic vesicles [@alfonso1993]. Null mutations of these genes are lethal due to the inhibition of worm's locomotion and feeding and its eventual death due to starvation [@rand1989; @alfonso1993]. Polymorphic ChAT and VAChT mutants in which the expression is reduced, but not abolished, revealed somewhat opposite phenotype. The pharyngeal pumping both in the presence and absence of food was reduced [@dalliere2015] the movement highly uncoordinated and jerky [@rand1984], whereas egg-laying increased [@bany2003].
Aldicarb is a synthetic carbamate mainly used as a nematicide (compound used to kill plant parasitic nematodes) [@lue1984] in the pest management system. Its mode of action is via inhibition of the acetylcholine esterase (AChE, the enzyme that breakdown acetylcholine released to the synaptic cleft) [@johnson1983]. When applied on worms, aldicarb causes hypercontraction of the body wall muscle ,leading to paralysis [@nguyen1995; @mulcahy2013], hypercontraction of the pharyngeal muscle and inhibition of feeding [@nguyen1995] as well as the inhibition of egg-laying [@nguyen1995]. These observations in conjunction with the phenotypical analysis of *cha-1* and *unc-17* mutants, suggest acetylcholine stimulates feeding, coordinates locomotion and inhibits egg-laying in *C. elegans*.
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(ref:cholsynapsecelegand) **Enzymes and transporters at the *C. elegans* cholinergic synapse.** Upon release into the synaptic cleft, acetylcholine is broken down to choline and acetate by acetylcholinesterase (AChE). Choline is taken up to the pre-synapse by a choline transporter (ChT). The acetyl group in transferred onto choline to product acetylcholine; a reaction catalysed by choline transferase (ChAT). Generated acetylcholine is pumped back into the synaptic vesicle by the vesicular acetylcholine transporter (AChT) for re-cycling. Names of genes are depicted in small blue letters. Image taken from @rand2006.
<!-- (ref:cholsynapsecelegand) **Enzymes and transporters at the *C. elegans* cholinergic synapse.** Upon release into the synaptic cleft, acetylcholine is broken down to choline and acetate by acetylcholinesterase (AChE). Choline is taken up to the pre-synapse by a choline transporter (ChT). The acetyl group in transferred onto choline to product acetylcho