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add chapter one, figures and references

parent d2dbc0ac
# General introduction {#chapter-1}
## Historical perspective of selective toxicity of neonicotinoids
Insecticides are compounds utilised in agriculture, medicine, industry and private households to control insect pests. One of the major factors influencing their commercial use is their ability to selectively act on the target species. The idea and importance of selectivity has been developed throughout the history of insecticide use which reaches back to 25th century BC.
Organic, natural compounds, contained within the plant or animal matter were in use until late 1800s [@agarwal2006]. For example, tobacco plant containing nicotine was applied in France to protect pear orchards against bug lice, whereas *Chrysanthemum* plants containing pyrethrum were used against worms and insects in America and Europe. In 1880s inorganic insecticides became popular, most notably arsenic-based compounds. Green pigment Paris green (cooper acetoarsenate) was used in controlling Colorado potato beetles and mosquitoes @cullen2008; @peryea1998], whereas lead arsenate was an effective insecticide for apple and cherry orchards [@peryea1998]. Although useful in agriculture, these compounds were toxic to humans. Acute exposure to high concentrations of arsenic increases lung cancer risk [@nelson1973], whereas ingestion of low doses of arsenic over a long time period can have several potential adverse effects [@gibb2010; @argos2010]. Due to these concerns, the use of lead arsenate has been effectively eliminated. However, because of the pesticide’s environmental persistence, it is estimated that millions of acres of land are still contaminated with lead arsenate residues. This presents a potentially significant public health concern in some areas of the United States (e.g., New Jersey, Washington, and Wisconsin), where large areas of land used historically as orchards have been converted into residential developments [@hood2006].
Many agrochemicals introduced into the market in the last century are synthetic and have improved safety profile. For example, DTT is generally considered safe to humans, however, it opposes a threat to the wildlife due to its propensity to bio-accumulate in the adipose tissues and persist in the environment [@EUEPA1975]. Insecticides organophosphates and carbamates are the cause of some serious human poisonings, some of which can lead to death. The lack of selectivity combined with emerging resistance instigated new management strategies aimed to combat these negative effects. In the 1990s research activities concentrated on finding new insecticides which have greater selectivity and better environmental and toxicological profiles.
<!-- Since the begining of 19th century, human population has been increasing exponatially, enhancing pressure on land to produce crops more efficiently. Since up to 90 % of the crops can be lost to pest-infestation usage of insecticides became an inseparable part of the agriculture. This is reflected in the -->
<!-- Some modern uses of arsenic-based pesticides still exist. Chromated copper arsenate (CCA) has been registered for use in the United States since the 1940s as a wood preservative, protecting wood from insects and microbial agents. In 2003, CCA manufacturers instituted a voluntary recall of residential uses of CCA-treated wood. CCA is still approved for use in nonresidential applications, such as in marine facilities (pilings and structures), utility poles, and sand highway structures (U.S. EPA, 2008; WPSC, 2008). -->
<!-- The use of organic arsenical pesticides began in the 1950s and has continued into the present day. Overall, organic arsenicals in the pentavalent oxidation state are much less toxic than inorganic arsenicals because, unlike inorganic arsenic, these ingested organic arsenicals are not readily taken up into cells and undergo limited metabolism (Cohen et al., 2006). Key pesticides based on organic arsenicals include monosodium methanearsonate (MSMAsV) and dimethylarsinic acid (DMAsV), also known as cacodylic acid. The use of these compounds as pesticides has given scientists the opportunity to comparatively study organic and inorganic arsenic toxicity and carcinogenicity, including investigations on metabolites of organic and inorganic arsenic compounds (e.g., the trivalent methylated arsenic compounds). In particular, the study of cacodylic acid has helped elucidate a potential mode of carcinogenic action for arsenic. In light of this, the U.S. Environmental Protection Agency (EPA) (2006) chose not evaluate DMAsV as carcinogenic to humans in the reregistration decision for this pesticide (U.S. EPA, 2006). Similarly, in 2007, a U.S. EPA Science Advisory Board (SAB) determined that DMAsV had the potential to cause cancer in humans only when doses are high enough to cause bladder cell cytotoxicity (U.S. EPA, 2007). Some uses of these compounds as herbicides were discontinued in 2009 (U.S. EPA, 2009). At that time, EPA reregistered MSMAsV for use on cotton but decided to stop the use of it on golf courses, sod farms, and right-of-ways in 2013. However, EPA agreed to reevaluate new information on the carcinogenicity of inorganic arsenic, the environmental degradation product of the organic arsenical products in 2012. EPA will consider the results of the reevaluation in make decisions regarding new applications for registration of MSMAsV. -->
## Development of neonicotinoid insecticides
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 substantially absorb in the sunlight, resulting in degradation. 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 was replaced by nitroguanidine and cyanoamidine [@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 EU in 2008.
(ref:neonics-structure) **Development and chemical structures of synthetic insecticides neonicotinoids.** Systematic modification of the lead and prototype compounds led to the discovery of seven neonicotinoids currently used in agriculture and animal health. They are classified according to the pharmacophore moiety into N-nitroguanidines, N-cyanoamidines and nitromethylenes.
```{r neonics-structure-label, fig.cap="(ref:neonics-structure)", fig.scap='Development and chemical structures of synthetic insecticides neonicotinoids.',fig.align='center', out.height = '60%', echo = FALSE}
knitr::include_graphics("fig/general_intro/png/neonics_structure.png")
```
## Physicochemical properties
Neonicotinoids exhibit beneficial physical and chemical property profile (Table \@ref(tab:properties)), therefore are stable in the environment and can be applied by a diversity of methods. Due to relatively high water solubility, they act as systemic insecticides [@westwood1998]. This means that once applied on crops, neonicotinoids dissolve in the available water and can be taken up by the developing roots or leaves. Upon plant entry, they are then distributed to all parts of the plant [@haas2006], providing protection against herbivorous pests. This systemic property of neonicotinoids means they can be used as a seed coating, reducing the required frequency of application. Indeed, seed dressing is the most commonly used method, accounting for 60 % of all neonicotinoids applications worldwide [@jeschke2011] and particularly popular to protect potatoes, oilseed rape, cereal, sunflower and sugar beet. In addition, neonicotinoids half-life in soil is from several weeks to years [@cox1997; @sarkar2001; @gupta2007), hence seed-dressing creates a continual source for re-uptake by plants. They are also suitable for ground treatment and are used as soil drenching for the protection of citrus trees and vines, granules for amenity grassland and ornament flowers and as a trunk-injection to protect trees against herbivores. Low vapor pressure and low Henry’s Law constant of neonicotinoids means they are not volatile, therefore can be applied as spray. This method is used in garden for flowers and vegetables and in agriculture on soft fruits and greenhouse crops. Low lipophilicity (indicated by octanol/water partition coefficient value (log Pow)) suggest they do not bio-accumulate in the adipose tissues [@turaga2016]. However, moderate water solubility combined with low lipophilicity means they may have a potential to accumulate in water.
Although structurally related nicotine has similar properties, it is not appropriate for the agricultural use due to low toxicity to insects [@nauen1996].
```{r properties, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
properties <- data.frame(
Compound = c("Nitenapyram", "Clothianidin", "Thiacloprid"),
log = c("-0.64 (1)", "0.70 (1)", "1.26 (1)"),
pKa = c("3.1 and 11.5", "11.09 (5)", "NA (5)"),
Water = c("590 000 (3)", "340 (3)", "184 (3)"),
Henry = c("4 x 10\\textsuperscript{-13} (5)", "3 x 10\\textsuperscript{-11} (5)", "5 x 10\\textsuperscript{-10} (5)" ),
Water = c("NA (3)", "56.4 (3)", "28.0 (3)"))
properties %>%
mutate_all(linebreak) %>%
kable("latex", align = "l", booktabs = TRUE, escape = F,
col.names = linebreak(c("Compound", "log Pow\npH=7.4\n24 $^\\circ$C", "pKa at\n20 $^\\circ$C", "Water solubility\nmg / L\n20 $^\\circ$C\npH=7", "Henry's law\nPa x m$^3$ x mol$^-1$\n20 $^\\circ$C", "Water sediment \nDT50 (days)")),
caption = 'Physichochemical properties of neonicotinoids',
) %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote("log Pow = octanol/water particioning, DT50 = half-life for degradation, 1 = Jeschke and Nauen 2008, 2 = Sangster 1997, 3 = Bonmatin et al., 2015, 4 = Maeda et al., 1978, 5 = Pesticide Properties Database (PPDB), 2019",
threeparttable = T)
```
<!-- ```{r properties, echo=FALSE} -->
<!-- library(kableExtra) -->
<!-- library(dplyr) -->
<!-- properties <- data.frame( -->
<!-- Compound = c("Nicotine", "Nitenapyram", "Clothianidin", "Thiacloprid"), -->
<!-- log = c("0.17 (5)", "-0.64 (1)", "0.70 (1)", "1.26 (1)"), -->
<!-- pKa = c("3.1 and 8.0", "3.1 and 11.5", "11.09 (5)", "NA (5)"), -->
<!-- Water = c("50 (5)", "590 000 (3)", "340 (3)", "184 (3)"), -->
<!-- Henry = c("3 x 10\\textsuperscript{-9} (4)", "4 x 10\\textsuperscript{-13} (5)", "3 x 10\\textsuperscript{-11} (5)", "5 x 10\\textsuperscript{-10} (5)" ), -->
<!-- Water = c("14.8 (2)", "NA (3)", "56.4 (3)", "28.0 (3)")) -->
<!-- properties %>% -->
<!-- mutate_all(linebreak) %>% -->
<!-- kable("latex", align = "l", escape = F, -->
<!-- col.names = linebreak(c("Compound", "log Pow\npH=7.4\n24 $^\\circ$C", "pKa at\n20 $^\\circ$C", "Water solubility\nmg / L\n20 $^\\circ$C\npH=7", "Henry's law\nPa x m$^3$ x mol$^-1$\n20 $^\\circ$C", "Water sediment \nDT50 (days)")), -->
<!-- caption = 'Physichochemical properties of neonicotinoids', -->
<!-- ) %>% -->
<!-- kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>% -->
<!-- footnote(general= "log Pow = octanol/water particioning, DT50 = , 1 = Jeschke and Nauen, 2008, 2 = Sangster, 1997, 3 = Bonmatin et al., 2015, 4 = Maeda et al., 1978, 5 = Pesticide Properties Database (PPDB), 2019", -->
<!-- threeparttable = T) -->
<!-- ``` -->
\newpage
<!-- Neonicotinoids are affective at sub-$\mu$M concentrations against piercing-sucking pest infestation such as aphids, whiteflies, thrips and planthoppers (Elbert et al., 2008). They also have a good safety profile. Toxicity is typically reported as LD50 (the amount of substance ingested that kills 50% of the population) or LC50 (the amount of substance in the surrounding medium needed to kill 50% of the population) and expressed in ng of substance/mg of body weight. LD50 of neonicotinoids against target species is typically lower than ng/mg (Beketov and Liess, 2008, Alexander et al., 2007), and at least 10 times higher for non-target insect species, such as honey bees (Apis mellifera) (Iwasa et al., 2004) and several hundred fold higher for birds and fish (De Cant, 2010). Mammals are the least susceptible with LD50 doses higher than 130 mg/kg of body weight (Legocki, 2008). When exposed topically, neonicotinoids exhibit sub $\mu$M LC50 against target species (typically between single to 2-digit ng/mg) (Lucas et al., 2004, De Cant, 2010, Mota-Sanchez et al., 2006, Zewen et al., 2003), and again at least 10-fold higher against non-target species (De Cant, 2010, Iwasa et al., 2004). -->
##Effects of neonicotinoids on target and non-taregt species ##{#nontargeteffect}
The diversity of neonicotinoid application methods, means that most environment types are exposed to these compounds. Therefore, a thorough understanding of their toxicity on a diversity of species is needed to ensure their use is safe to humans and the environment. Toxicity of compounds is typically reported as LD50 (the amount of substance ingested that kills 50 % of the population) or in the LC50 (the concentration of substance in the surrounding medium that kills 50 % of the population). The assessments are performed following short term or long-term exposure, upon topical or oral applications.
<!-- Lethality of neonicotinoids depends on many factors, including a compound of interest, time and route of exposure. Generally, oral exposure is more toxic than contact exposure, whereas nitro- are come toxic than cyano- neonicotinoids, possibly due to their enhanced metabolism [@suchail2004; @brunet2005]. -->
###Insect-pests
<!-- look at this paper to see the symptoms of imi exposure on insects -->
<!-- @sone1994 -->
Toxicity assessments on mammals, fish, insects and nematodes revealed insects as the most susceptible taxon (Table ref). The LC50 concentration following both topical and oral exposure varies between
2.54 nM on mayfly (*E. longimanus*) to 3.87 $\mu$M on aphid *M. persicae*. The susceptibility depends on the route of exposure. Imidacloprid applied topically onto the abdomen of *M. persicae* at 1.83 $\mu$M kills 50 % of the population. This increased by over 6-fold upon ingestion of this compound. The sensitivity of insects to neonicotinoids also increases upon extended exposure time. The LC50 of imidacloprid on Mayfly increases from 82.13 nM to 2.54 nM after 24- and 96- hour treatment, respectively [@alexander2007].
The LD50 of neonicotinoids varies from 8.8 to 59 ng/pest. This could be partially accounted for by the differences in the body weight of these animals. The Colorado potato beetle, *L. decemlineata* weights approximately 175 mg, therefore the LD50 of neonicotinoids on this species is between 0.05 and 0.5 ng / mg of body weight. Whereas the LD50 of neonicotinids on *N. lugens'* average body weight is 1 mg, therefore the LD50 of neonicotinoidson this species expressed in ng/mg is 0.82 [@zewen2003].
Interestingly, although nicotine is structural related to neonicotinoids, it is much less potent at eradicating pests. The LC50 and LD50 values for nicotine are at least 6-fold higher than those for neonicotinoids, highlighting its unsuitability for agricultural use. Concentrations between of 19-38 $\mu$M in the plant sap are generally considered sufficient to protect plants against insect-pests [@byrne2006; @castle2005].
<!-- [@zewen2003] microtopical application Nilaparvata lugens -->
<!-- 0.82 ng / pest -->
<!-- Myzus [@bass2011]: again topical aplication onto dorsal abdomen named The 'micro-application' bioassay -->
<!-- Also spray appplication . Assesed 72 hours later The 'spray application' bioassay -->
<!-- Imi and thiame 0. 99 and 0 .64 mg / L in topical application -->
<!-- Spray application : 0.14 and 0.48 mg / L. -->
<!-- oral : sachet test in which aphid feeds on treatment containing liquid sucrose solution -->
<!-- Below taken from @mota-sanchez2006 -->
<!-- Atopical aplication in which a small quantity of the treatment in applied onto the dorsal thorax of the animal. Toxicity assesed 72 hours later. The Colorado potato beetle, Leptinotarsa decemlineata - weight 150 - 200 mg. LD50 from 0.024 (acetamidprid) to 0.88 for dinotefuran. Tested Thia, Imi, Nit, Thia, Clo. Nicotine 61.3 ug / beetle. Calculated per mg of body weight (taking average body weight of 175 mg) = . -->
<!-- Injection studies: treatment injected into the abdomen. LD 50 0.024 ug / beetle for acetamidprid to 0. 088 for dino . Nicotine 61 -->
<!-- [@nauen1996] M.persicae -->
<!-- in dip test -->
<!-- imi = 0.47 ppm -->
<!-- nicotine = >300 ppm -->
<!-- in sachet test (oral exposure) : -->
<!-- imi 0.073 ppm -->
<!-- nicotine 4.5 ppm -->
<!-- Ref 1 = @nauen1996 -->
<!-- Ref 2 = @mota-sanchez2006 -->
<!-- Ref 3 = @bass2011 -->
<!-- Ref 4 = @zewen2003 -->
<!-- Ref 5 = - reported in @tomizawa2000 -->
<!-- Ref 6 = @alexander2007 -->
<!-- Ref 7 = @decant2010 -->
<!-- Ref 8 = @luo1999 -->
<!-- Ref 9 = @decant2010 -->
<!-- Ref 10 = @wang2012 -->
<!-- Ref 11 = @wang2015 -->
<!-- Ref 13 = @dong2017 -->
<!-- REf 14 = @cresswell2011 -->
<!-- Ref 15 = @godfray2015 -->
<!-- ```{r } -->
<!-- library(kableExtra) -->
<!-- library(dplyr) -->
<!-- toxic <-data.frame ( -->
<!-- Drug = c("Imi", "Imi", "Nic", "Nic", "Imi", "Thtx","Imi", "Thtx","Nic", "Dino", "Thia", "Imi", "Nit", "Thia", "Clo", "Ace", "Imi", "Thia", "Nic", "Imi", "Imi", "Imi", "Imi", "Imi", "Clo", "Thx", "Clo", "Clo", "Clo", "Clo", "Clo", "Clo", "Imi", "Imi", "Clo", "Clo", "Clo", "Imi", "Acet", "Nit", "Clo", "Thia", "Thia"), -->
<!-- Species = c("M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae","M.persicae", "M.persicae", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "N.lugens","M. domestica", "M. domestica", "E. longimanus", "E. longimanus", "A. mellifera", "A. mellifera", "A. mellifera","A. mellifera","A. mellifera", "Bobwhite quail", "Bobwhite quail", "Mallard duck", "Mouse", "O. mykiss", "L. macrochirus", " E. fetida", "E. fetida", "E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", "M. incognita"), -->
<!-- Taxon = c("Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect","Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "insect", "Insect", "Insect", "Bird", "Bird", "Bird", "Mammal", "Fish", "Fish", "Earth worm", "Earth worm","Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Nematode"), -->
<!-- LD50 = c("-", "-", "-", "-", "-", "-", "-", "-", "61.3 ng /beetle", "8.8 ng / beetle", "6.8 ng/beetle", "59 ng/beetle", "35 ng/beetle", "32 ng/beetle", "27 ng/beetle", "24 ng/beetle", "0.82 ng/ pest", "3 ng/mg", ">50 ng/mg", "-", "-", "-", "4.5 ng/ bee", "3.8 ng/bee", "3.7 ng/bee", "2.4 ng/bee", ">200 mg/kg (acute)", ">5040 mg/kg (5 days)", ">5230 mg/kg (5 days)", "389-465 mg/kg", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-"), -->
<!-- LC50 = c("0.47 mg/L", "0.073 mg/L", ">300 mg/L", " 4.5 mg/L", "0.99 mg/L", "0.64 mg/L", "0.14 mg/L", "0.48 mg/L", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "0.021 mg/L (24 hrs", "0.00065 mg/L (96 hrs", "1.76 mg/L", "-", "-","-","-","-", "-", "-", "-", "106 mg/L", "117 mg/L", "1.23 mg/L (24 hours)", "0.70 mg/L (48 hours)", "15. 5 mg / L (14 days", "7.44 mg/L (7 days)", "6.06 mg/L (14 days)", "3.05 mg/L (14 days)", "2.69 mg/L (14 days)", "4.34 mg/L (14 days)", "0.93 mg / L (14 days)", "2.68 mg / L (14 days)", "36.2 mg / L (6 hours)"), -->
<!-- Bioassay = c("Topical", "Oral", "Topical", "Oral", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "?", "?", "Topical", "Topical", "oral", "oral", "oral", "oral", "oral", "oral","oral", "oral", "oral", "?", "?", "topical", "topical", "topical", "topical", "topical", "topical", "topical", "topical", "topical", "topical", "topical"), -->
<!-- Ref = c("1", "1", "1", "1", "3", "3", "3", "3", "2", "2", "2", "2", "2", "2", "2", "2", "4", "5", "5", "6", "6", "14", "14", "15", "15", "15", "7", "7", "7", "7", "7", "7", "8", "8", "9", "10", "10", "11", "11", "11", "11", "12", "13")) -->
<!-- toxic %>% -->
<!-- kable("latex", align = "l", escape = F, -->
<!-- caption = 'Toxicity') %>% -->
<!-- kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>% -->
<!-- footnote (general = " References (Ref) 1: Nauen et al. [1996], 2: Mota-Sanchez et al. [2006], 3: Bass et al. [2011], 4: Zewen et al. [2003], 5: reported in Tomizawa et al. [2000], 6: Alexander et al. [2007], 7: De Cant and Barrett [2010], 8: Luo et al. [1999], 9: De Cant and Barrett [2010], 10: Wang et al. [2012], 11: Wang et al. [2015], 13: = Dong et al. [2017], 14: Cresswell [2011], 15: = Godfray et al. [2015]") -->
<!-- ``` -->
<!-- Ref 1 = @nauen1996 -->
<!-- Ref 2 = @mota-sanchez2006 -->
<!-- Ref 3 = @bass2011 -->
<!-- Ref 4 = @zewen2003 -->
<!-- Ref 5 = - reported in @tomizawa2000 -->
<!-- Ref 6 = @alexander2007 -->
<!-- Ref 7 = @decant2010 -->
<!-- Ref 8 = @luo1999 -->
<!-- Ref 9 = @decant2010 -->
<!-- Ref 10 = @wang2012 -->
<!-- Ref 11 = @wang2015 -->
<!-- Ref 13 = @dong2017 -->
<!-- REf 14 = @cresswell2011 -->
<!-- Ref 15 = @godfray2015 -->
\newpage
```{r echo=FALSE, warning = FALSE, message = FALSE}
library(kableExtra)
library(dplyr)
toxic <-data.frame (
Drug = c("Imi", "Imi", "Nic", "Nic", "Imi", "Thtx","Imi", "Thtx","Nic", "Dino", "Thia", "Imi", "Nit", "Thia", "Clo", "Ace", "Imi", "Thia", "Nic", "Imi", "Imi", "Imi", "Imi", "Imi", "Clo", "Thx", "Clo", "Clo", "Clo", "Clo", "Clo", "Clo", "Imi", "Imi", "Clo", "Clo", "Clo", "Imi", "Acet", "Nit", "Clo", "Thia", "Thia"),
Species = c("M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae","M.persicae", "M.persicae", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "N.lugens","M. domestica", "M. domestica", "E. longimanus", "E. longimanus", "A. mellifera", "A. mellifera", "A. mellifera","A. mellifera","A. mellifera", "Bobwhite quail", "Bobwhite quail", "Mallard duck", "Mouse", "O. mykiss", "L. macrochirus", " E. fetida", "E. fetida", "E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", "M. incognita"),
Taxon = c("Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect","Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "insect", "Insect", "Insect", "Bird", "Bird", "Bird", "Mammal", "Fish", "Fish", "Earth worm", "Earth worm","Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Nematode"),
LD50 = c("-", "-", "-", "-", "-", "-", "-", "-", "61.3 ng /beetle", "8.8 ng / beetle", "6.8 ng/beetle", "59 ng/beetle", "35 ng/beetle", "32 ng/beetle", "27 ng/beetle", "24 ng/beetle", "0.82 ng/ pest", "3 ng/mg", ">50 ng/mg", "-", "-", "-", "4.5 ng/ bee", "3.8 ng/bee", "3.7 ng/bee", "2.4 ng/bee", ">200 mg/kg (acute)", ">5040 mg/kg (5 days)", ">5230 mg/kg (5 days)", "389-465 mg/kg", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-"),
LC50 = c("1.83 $\\mu$M", "285.49 nM", "1.85 mM", "27.74 mM", "3.87 $\\mu$M", "2.19 $\\mu$M", "257.52 nM", "1.64 mg/L", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "82.13 nM (24 hrs)", "2.54 nM (96 hrs)", "6.88 $\\mu$M", "-", "-","-","-","-", "-", "-", "-", "424.51 $\\mu$M", "468.60 $\\mu$M", "4.81 $\\mu$M (24 hours)", "2.74 $\\mu$M (48 hours)", "62.08 $\\mu$M (14 days)", "24.24 $\\mu$M (7 days)", "24.27 $\\mu$M (14 days)", "11.93 $\\mu$M (14 days)", "12.08 $\\mu$M (14 days)", "26.75 $\\mu$M (14 days)", "3.72 $\\mu$M (14 days)", "10.60 $\\mu$M (14 days)", "143. 24 $\\mu$M (6 hours)"),
Bioassay = c("Topical", "Oral", "Topical", "Oral", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "?", "?", "Topical", "Topical", "Oral", "Oral", "Oral", "Oral", "Oral", "Oral","Oral", "Oral", "Oral", "?", "?", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical"),
Ref = c("1", "1", "1", "1", "3", "3", "3", "3", "2", "2", "2", "2", "2", "2", "2", "2", "4", "5", "5", "6", "6", "14", "14", "15", "15", "15", "7", "7", "7", "7", "7", "7", "8", "8", "9", "10", "10", "11", "11", "11", "11", "12", "13"))
toxic %>%
kable("latex", align = "l", escape = F, booktabs = TRUE, longtable = TRUE,
caption = 'Toxicity of nicotine and neonicotinoids') %>%
kable_styling(font_size=10, position = "center", full_width = FALSE, latex_options = c( "hold_position", "repeat_header")) %>%
add_footnote ("References (Ref) 1: Nauen et al. 1996, 2: Mota-Sanchez et al. 2006, 3: Bass et al. 2011, 4: Zewen et al. 2003, 5: reported in Tomizawa et al. 2000, 6: Alexander et al. 2007, 7: De Cant and Barrett 2010, 8: Luo et al. 1999, 9: De Cant and Barrett 2010, 10: Wang et al. 2012, 11: Wang et al. 2015, 13: = Dong et al. 2017, 14: Cresswell 2011, 15: = Godfray et al. 2015")
```
<!-- The LC50 values vary from sub $\mu$g / L to 73 $\mu$g / L [reviewed in @goulson2013]. For example, the LC50 of imidacloprid on peach aphid *Myzus persicae* and housefly *Musca domestica* is 73 $\mu$g / L [@nauen1996] and 45 $\mu$g / L [@tomizawa2000], respectively. The LC50 of imidacloprid on the mayfly is lower and dependent on the exposure. After 24 hour exposure, it is 2.1 $\mu$g / L and 0.65 $\mu$g / L after 3-day exposure [@alexander2007]. -->
Persistent use of neonicotinoids leads to the development of resistance [@liu2006]. Neonicotinoid-resistant strain of *M. persicae* was isolated from the peach orchard fields in France [@bass2011]. Its susceptibility to imidacloprid was over 1500-fold lower than the susceptibility of the non-resistant clone, as indicated by the shift in the LC50 [@bass2011]. This strain originally isolated in 2011, has spread to Southern Europe and Northern Africa [@charaabi2018], where it may account for as much as 55 % of all aphids [@charaabi2018].
Resistant strain of the Colorado potato beetle in the United States was also isolated [@szendrei2012]. The development of resistance and the rapid expansion of resistant strains opposes a serious threat to the successful control of pests in the agriculture.
### Mammals, fish and birds
Toxicity of neonicotinoids to mammals and fish is low in comparison to insect pests. The LC50 of clothianidin on Mallard duck and bobwhite quail is over 424 $\mu$M [@decant2010] following oral application for 5 days. The LD50 of imidacloprid on mammals and fish varies greatly but it is generally over 10 mg/kg of body weight and can be as high as 2000 mg/kg of body weight.
Evidence suggests that environmental relevant concentrations of neonicotinoids may have negative effects on birds [@hallmann2014]. In particular, granivorous and insectivorous vertebrates may be at risk, should they consume neonicotinoid-contaminated seeds and/or insects [@goulson2013]. Environmental neonicotinoids may induce lethal [@lopez-antia2013] but also sublethal effects on birds such as impaired migratory ability [@eng2017], impaired growth and reproduction [@sanchez-bayo2016].
<!-- Imidacloprid and acetamiprid may have an effect on mammalian spert fertilisation in mouse [@gu2013] -->
### Soil worms
<!-- Note that 1 mg / kg of soil = 1 ppm = 1 mg / L -->
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].
The toxicity of five neonicotinoids were tested on the mortality of *Eisenia fetida* [@wang2015; @wang2012]. The order of potency of these compounds is: clothianidin > thiacloprid ∼ acetamiprid ∼ imidacloprid > nitenpyramm. The LC50 of the most and least potent compounds are 3.72 $\mu$M and 26.75 $\mu$M, respectively. Other soil worms may be less susceptible than *E. fetida*. Clothianidin at $\ge$ 400 $\mu$M, had no clear effect on mortality of the common earthworm *Lumbricus terrestris* (*L. terrestris*) [@basley2017]. However, neonicotinoids may have an antiparasitic potential. Thiacloprid eradicates juveniles of plant parasitic nematode *Meloidogyne incognita* (*M. incognita*) at LC50 of 143.24 $\mu$M [@dong2017].
Investigations into the sublethal effects of neonicotinoids on earthworms, typically following 7 or 14 day treatment period were also identified. Cocoon production of *E. fetida* was reduced by clothianidin and thiacloprid at concentrations $\ge$ than 1.2 $\mu$M; the EC50 of 5.1 $\mu$M and 3.4 $\mu$M, respectively were reported [@gomez-eyles2009]. Fertility of other worm species in response to neonicotinoids was also observed, including *Lumbricus rubellus* (*L. rubellus*) [@baylay2012], *Dendrobaena octaedra* (*D. octaedra*) [@kreutzweiser2008] and *Eisenia andrei* [@alves2013]. Reduction of body weight of *E. fetida* and *D. octaedra* was observed following after 14-day treatment with imidacloprid at 27.08 and 54.75 $\mu$M [@kreutzweiser2008]. Imidacloprid at 488.85 nM to 7.82 $\mu$M increased avoidance of *E. andrei* [@alves2013] and at 782 nM, it reduced the *A. caliginosa* burrowing depth and length [@dittbrenner2011]. Burrowing of *L. terrestris* was also impacted, but at higher imidacloprid concentrations [@dittbrenner2011]. 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]. Acute exposure to thiacloprid at 37 nM has an effect on chemosensing, whereas at 18 $\mu$M it impaired motility of this worm [@hopewell2017]. Impaired motility of *C. elegans* in response to $\ge$ 120 $\mu$M imidacloprid was also recorded [@mugova2018].
The risk opposed by the neonicotinoids depends on the time- and route of exposure, the innate sensitivity of worms, type of neonicotinoid present, the concentrations and their persistence in soil.
The concentration of neonicotinoids in soils has been estimated in several studies. @jones2014 collected soils from English fields prior to planting. In the center of the field, clothianidin concentration ranged from 80.10 nM to 54.47 $\mu$M for clothianidin, from less than 23.01 nM to 41.84 $\mu$M for imidacloprid and from 68.56 to 5.14 $\mu$M for thiamethoxam. The detected neonicotinoid concentrations in the field margins were generally lower. USA soil samples were collected from fields treated with clothianidin-coated seeds. Clothainidin concentration shortly after sowing was 44.06 $\mu$M. This decreased to below 8.01 $\mu$M 10 months later [@perre2015].
<!-- [@botias2015] -->
<!-- Selected 5 oilseed or wheat fields treated for at least three previous years with neonicotinoids. Soil samples were collected 10 months after sowing. The presence of neonicotinoids was detected using spectrometry. oilseed rape cropland : mean 3.46 ng / g of tmx, 13.28 clo, imc 3.03, thc 0.04 ng / g. -->
<!-- Margin : tmx 0.72 ng / g, clo 6.57, imc 1.92, thc $\le$ 0.01 and this is not significantly differen t from the margins of the wheat fields. -->
Based on laboratory conditions, half life of neonicotinoids is highly variable ranging from 8 days for nitenpyram to almost 7000 for clothianidin [reviewed in @bonmatin2015]. Several field studies were conducted to estimate dissipation time of neonicotinoids. @schaafsma2016 collected 18 Canadian and pre-planting soil samples over 2 successive years. These samples originated from variable-crop fields which had been treated for at least 4 successive years. Concentration of clothianidin and thiacloprid was measured in year 1 samples and averaged at 19.22 $\mu$M. Based on the history of crops planted and insecticide recharge regime, the TD50 of clothianidin was estimated at 0.4 years 9 (146 days). It was also concluded clothianidin and thiacloprid residues would accumulate for 3-4 years and plateau at ~ 23 $\mu$M in agricultural fields with one-year insecticide application routine. Using similar approach, @schaafsma2016 also estimated the DT50 of imidacloprid at 208 days.
Based on these data the average total neonicotinoid concentration is in the region of 20 $\mu$M. Therefore, average concentrations of neonicotinoids in the field are lower than doses causing worm lethality. However, higher concentrations of neonicotinoids may be experienced, particularly during the sowing period.
It is also probable that the concentration of neonicotinods is higher near the coated seed. The coating typically contains from 0.17 - 1 mg of the active ingredient [@goulson2013]. Up to 98 % of the active ingredient leaches into the environment [@goulson2014]. Therefore upper concentrations of environmental doses may have sublethal effect on soil inhabiting worms and nematodes.
<!-- Based on these data, neonicotinoids may accumulate in soil for several years, exposing residing worms for -->
<!-- FIELD STUDIES: -->
<!-- [@schaafsma2016] note that he estimated the DT50 based on 8 year history of crops and typical recharge regime. -->
<!-- Soil has been treated with neonics for the previously treated for at least 4 years with neonics once a year in a form of seed dressing. 18 samples collected from Canada fields on which various crops were grown. These were collected before planting. Average measured conc of clo and thia = 4.8 ng / g. Repeated mesurement one year after and estimated half life of 0.4 years. Based on 8 year crop history it was concluded that the residues of neonics would accumulate for 3-4 years and plateou at 6 ng / g in arricultural crops ontowhich one-year insecticide application routine followed. Also calculated TD50 for imidacloprid in the same way and 0.57 years. -->
<!-- [@xu2016] here actual data was obtained from field studies -->
<!-- 50 samples collected over 2 year period from US corn fields which following 1-yearly regime of clothianidin applicatioin for in at least 2 and for max of 11 preeceding years. Samples collected shortly after planting and neonic app. At the tie of sampling average conc of clothianidin = 6.4 ng / g. -->
<!-- Following yearly recharge regime, concentrations increase over time for 5-4 years and plateou after that. -->
<!-- Imidacloprid : based on laboratory data, hald life of imidacloprid varies between 148 and 7000 days [@decant2010]. -->
<!-- Amount of neonicotinoids active against earthworms and nematodes are much higher than the typical field-realistic concentrations. In waters, neonicotinoids are typically present at below 0.1 $\mu$g / L, although some studies report concentrations as high as 320 $\mu$g / L (reviewed in (Francisco Sánchez-Bayo, 2016). In soils, the concentrations are typically below 10 ng / g of soil [@goulson2013]. -->
### Insect pollinators
<!-- LD50 on bees = single digit ng / insect (which is approx 0.1 g in weight) -->
Although insect pollinators includes many species of bees, moths and butterflies, the breadth of toxicological knowledge is derived from research involving honeybee *Apis mellifera*. The LD50 values of neonicotinoids are usually reported in g/bee. These values were multiplied by 10 to standardize units to ng/g of body weight. The LD50 of imidacloprid on honeybee is 4.5 ng/mg of body mass, whereas the LC50 is 7.04 $\mu$M. These values were derived by @cresswell2011, who pooled the results of 13 studies most of which reported the effects of acute oral toxicity. European Food Safety Authority investigated the toxicity of other neonicotinoids [@godfray2015]. The estimated acute oral toxicity of imidacloprid, clothianidin, thiamethoxam is 3.8, 3.7, and 5.0 ng/bee. The average weight of a bee is 100 mg, hence the LD50 expressed in ng/mg is 0.038, 0.037 and 0.05, respectively. whereas the acute contact toxicity is 81, 44 and 24 ng/mg of body weight. The acute contact toxicity are lower: 0.81, 0.44, 0.24 ng/mg of body weight, respectively. Based on data collected from 17 species, the susceptibility of different bee species to neonicotinoids varies, but only slightly [@spurgeon2016].
To determine whether environmental concentration of neonicotinoids are lethal to bees, the concentrations of neonicotinoids in nectar, wax and pollen was been investigated. Generally, the highest concentration are found in the former [@goulson2013]. @cresswell2011 determined that the average concentration of imidacloprid in most commonly bee-consumed nectar varies between 2.3-20 $\mu$M. He also estimated that the average realistic amount of imidacloprid in nectar load is 0.024-0.3 ng. In comparison to the LC50 and the LD50 doses, this is higher, therefore field-realistic concentration of neonicotinoids should not cause bee lethality. However, a substantial body of evidence from lab- and field- based experiments suggest that ecologically relevant doses of neonicotinoids impair on the cognitive function and reproduction of these biological pollinators.
Honeybees exposed to a single dose of thiamethoxam, clothianidin and imidacloprid exhibited reduced homing rates, and in case of thiamethoxam – increased lethality [@henry2012; @schneider2012]. Oral application of imidacloprid led to reduced ability to olfactory learn, and compromised foraging activity [@decourtye2004]. Increased home failure as well as reduced pollen foraging efficiency was also noted in bumblebees exposed chronically (14 days) to imidacloprid [@feltham2014]. Reduced response in conditioning assay was noted in honey bees treated with imidacloprid chronically (4 days), which were then allowed to recover for three days [@williamson2013]. In addition, 14-days exposure of bumblebees to imidacloprid increased number of empty pupal cells, reduced number of queens produced and total size of treated colonies [@whitehorn2012]. 4- week exposure of bumblebee early-developmental stages to imidacloprid reduced worker production, pollen foraging efficiency and duration. An increase in foragers’ recruitment and workers mortality was also reported [@gill2012]. Chronic oral exposure (weeks) of worker bumblebees to imidacloprid resulted in reduced fecundity [@laycock2012; @tasei2000] whereas exposure of drones to field-realistic thiamethoxam and clothianidin concentrations led to shortening of life-spam and hindered sperm vitality and quantity [@straub2016]. In field studies, bumblebees foraging on oilseed rape coated with clothianidin, exhibited decreased queen production, colony growth and reduced bumblebee density [@rundlof2015]. More recently, international field studies confirmed negative effects of neonicotinoids on overwinter success and reproduction of honey and wild bees [@woodcock2017].
Insect pollinators play an important ecological, economical and evolutionary role. They pollinate wild plants [@kwak1998], food crops [@klein2007] and promote plant sexual reproduction [@gervasi2017]. The emerging evidence of the negative impact of neonicotinoids on honeybees, restricted their use in Europe. This highlights the importance of selective toxicity of insecticides in successful pest management programmed. The development of new insecticides, effective against pest and not beneficial insects or other species requires a detailed knowledge of their mode of action. This includes a detailed description of the molecular and structural determinants of their action. Next chapters describes nicotinic acetylcholine receptors - the main side of action of neonicotinoids.
## Overview of nicotinic acetylcholine receptors
nAChRs are members of the pentameric ligand-gated ion channels, found in a diversity of species from bacteria to human. They are the representatives of the cys-loop super family of channels which also include $\gamma$ -aminobutyric acid type A (GABA) receptors, 5-hydroxytryptamine type-3 receptors (5-HT3), and glycine receptors. Name cys-loop was devoted because all members contain a disulphide bond between cystines separated by a highly conserved sequence of 13 amino acids. Cys-loop receptors also share a common topology (Figure \@ref(fig:nachr-topology-label)): five receptor subunits arranged around the central pore. The five subunits can be the same, or different and form homo- or hetero-pentamers.
(ref:nachr-topology) **Topology of cys-loop receptors.** A single subunit of the cys-loop receptor consists of 4 transmembrane subunits (M1 - M4) and a conserved disulphide bridge in the N-terminal extracellular domain. 5 subunits come together to form a functional receptor (b). Receptors can be formed from either 5 identical (c) or from a combination of different subunits (d).
```{r nachr-topology-label, fig.cap="(ref:nachr-topology)", echo=FALSE, fig.scap= 'Topology of cys-loop receptors.', fig.align='center', out.height = '80%', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/nAChR_topology_3.png")
```
\newpage
### Biogenesis {#biogenesis}
The process of nAChR formation is complicated and complex but can be summarised in several steps: (1) subunit synthesis and folding, (2) assembly of subunits into functional pentameric receptor, (3) trafficking and targeting of subunits to the plasma membrane and degradation and/or recycling (4) (Figure \@ref(fig:turnover-label)). The process starts in the nucleus where nAChR gene is transcribed into the mRNA. mRNA contains specific signal sequence which allows for nucleus exit and targeting to the endoplasmic reticulum (ER). Upon arrival at the ER membrane, the co-translational synthesis of receptor subunit occurs, starting from the N-terminus. The protein is inserted into the ER membrane, the signal sequence is cleaved off, and the N-glycosylation chain is attached to the glycosylation recognition sequence [@blount1990]. In addition, the initial folding and oligomerisation occurs. Upon completion of protein synthesis, post-translation events are taking place which include disulphide bridge formation [@blount1990] and further folding and oligomerisation. There are two models describing the process of subunit assembly based of mammalian muscle receptors. First assumes that fully folded subunit forms dimers, and then assembly into pentameric asemblies [@gu1991]. Second theory describes that trimers are formed before the remainder subunits are added to form a pentamer [@green1993].
The process of subunit assembly is complex and probably regulated by a set of rules, such as the control over the subunits expression in the cell [@missias1996] and the primary structure of receptor subunits. In particular sequences within N-terminal (extracellular ligand binding domain) [@sumikawa1992; @sumikawa1994; @kreienkamp1995] but also regions within the TM domain [@wang1996a] and C-terminus [@eertmoed1999]. During receptor assembly, these sequences become buried within the folding protein and the receptor can be transported to the cell surface. Should a protein misfold, the sequences become exposed and the protein retained in the ER. I
Assembly and oligomerisation requires a number of ER and Golgi resident chaperons. Resistant to inhibitors of cholinesterase (RIC)-3 transmembrane protein [@millar2008], important in this process [@williams2005; @lansdell2005]. There are also other chaperons including immunoglobulin heavy chain binding protein (BiP) [@gething1999], calnexin [@ellgaard2001], UNC-50 [@eimer2007] and NACHO [@gu2016]. ER lipid composition can also effect assembly and trafficking. For example, spingolipids are important in cellular trafficking of vertebrate muscle receptors [@baier2006]. Therefore, the process of receptor biogenesis is complex and highly regulated. Should a protein misfold or misoligomerise, it is sent for degradation [@brodsky1999] mediated by the ER-associated system [@hampton2002]. In contrast, properly folded and oligomerised receptors are targeted to appropriate cellular localisation. Neuronal receptors are sent to the synapse, those in muscle cells are targeted to the NMJ during synaptogenesis, where they can perform their important function in signal propagation. The process of receptor turnover is demostarted in Figure \@ref(fig:turnover-label).
(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")
```
### Function
nAChRs are important for the function of the nervous system. Simplistically, nervous system is a complex network of the supporting cells named glia, and the basic working cells called neurons. Neurons consist of three main parts: (1) the cell body with nucleus where the majority of proteins are synthesised (2) dendrites which integrate information received from other neurons and propagate signal across the cell and (3) axonal appendages which receive signals and transmit them to other neurons. The nervous system carries out complex functions including detection of external and internal stimuli, transmission and integration of information and formation of memory and plasticity [@cooke2006]. All these processes relay on trans-neuronal communication between cells based on signal propagation and transmission.
Signal propagation throughout the nervous system is mediated by means of electrical impulses. Neurons are electrically excitable due to electrochemical potential across plasma membrane. Simplistically, the concentration of potassium ions is higher inside in comparison to the outside of the cell and the reverse is true for sodium ions and chloride. As a result of differential ionic distribution there is a chemical gradient across the plasma membrane. In addition, there is a difference in net charge between the extracellular and intracellular environments. The inside is negatively charged in comparison to the outside, creating an electrical gradient. At rest, this is maintained by large by integral membrane pumps. Upon input from another cell, the ionic distribution shifts which leads to either excitation or inhibition of a neuron.
The process of signal propagation throughout the nervous system relays on trans-synaptic signal transmission occurring at synapses and mediated by a neurotransmitter. There are two types of synapses: chemical and electrical (also known as gap junctions). Cys-loop receptors are mediating the the function of the former. Synapse is a space where an axon meets a dendrite (alternatively muscle cells at the neuromuscular junction (NMJ)). Upon arrival of a signal, neurotransmitter is released from the presynaptic neuron to act on ligand gated ion channels present at the postsynaptic membrane. Upon neurotransmitter binding, these channels open and allow selective ion movement down the electrochemical gradient. Flow of cations leads to depolarisation of the cell (cell becomes more positive) and excitation, or muscle contraction at the NMJ [@hille1978]. In contrary, flow of anions leads to hyperpolarisation (cell become more negative) and inhibition of signalling. The signal is terminated mainly by neurotransmitter breakdown but also by its transport into the pre-synaptic terminal by integral membrane protein.
Neurotransmitters determine whether the signal is inhibitory or excitatory. Acetylcholine (ACh) is an example of an excitatory neurotransmitter. It functions both in vertebrates and invertebrates and mediates signalling in the nervous system and at the NMJ [@williamson2009]. Acetylcholine acting on the post-synaptic nAChRs propagates signal between cells. The release of ACh from pre-synapse and subsequent neurotransmitter binding to nAChRs leads flow of sodium, potassium and sometimes calcium [ref] leading to depolarisation of the post-synaptic cell (Figure \@ref(fig:cholineric-synapse-label)). ACh can also act on other class of receptors, the metabotropic G-protein coupled receptor, which modulates neurotransmission. The signal is terminated mainly by synaptic enzyme cholinesterase which hydrolyses ACh to choline and acetate [@fukuto1990], but also by choline uptake to the presynaptic cell by Na^+^-choline transporter.
(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.
```{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")
```
### Kinetic properties
Electrophysiological recordings from recombinant and native nAChRs have greatly advanced knowledge of the pharmacological and kinetic properties of these channels. A range of vertebrate (reviewed in @millar2009) and invertebrate nAChRs [@ballivet1996; @squire1995] have been studied. The currents recorded from the whole cells expressing nAChRs were recorded following an exposure to the endogenous or exhogenous compounds.
Acetylcholine is a natural neurotransmitter of nAChRs. Upon its application, the nAChR open and allow flow of ions down their concentration gradient. nAChRs conduct cations, potassium and sodium and sometimes also calcium. For example, mammalian $\alpha7$ homopentamers are highly permeable to calcium, but other receptors exhibit low calcium permeability [@fucile2004]. Continuous application of the agonist, leads to receptor desensitisation [@katz1957]. Desensitized receptor adopts a distinct conformation in which an agonist is tightly bound, but the receptor is no longer capable of ion conduction [@nemecz2016]. This phenomenon is very rapid and varies between receptors from under a millisecond [@bouzat2008] to hundreds of milliseconds [@gerzanich1994] following the agonist application. Recovery from desensitisation is receptor-, compound- [@briggs1998] and concentration- dependent [@gerzanich1994]. After removal of the agonist, the receptor is capable of eliciting next response within a few seconds [@bouzat2008]. However, full recovery may not occur or may be slower if the receptor are exposed to the large doses of agonist for a prolonged time period [@katz1957].
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 noting event. In steed, 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].
### Diversity
The function of ACh on ligand-gated ion channels is mediated by a diversity of receptor subunits. Insect and vertebrates typically express ~10 nAChR subunits, whereas nematode *C. elegans* staggering 29 subunits. These subunits can be broadly divided into $\alpha$ and non-$\alpha$ depending on the presence or absence of the disulphide bond between the adjacent cystines in the extracellular N-terminal domains.
#### Insects
Acetylcholine is a major neurotransmitter in the brain of insects [@florey1963] whereas nAChRs are involved in formation of memory and sensory processing.
nChRs are expressed pre-post-synaptically at the soma, as determined mainly with electrophysiological approaches, whereby upon application of classical agonists, nAChR-like currents were elixited from neuronal preparations; these currents were blocked with nAChR antagonists. The presence of nAChRs in the Kenyon cells was shown by recording the spontaneous and nicotine or acetylcholine-evoked post-synaptic currents of neuronal preparations extracted from Drosophila. These responses were blocked by selective nAChR antagonists $\alpha$-Bgtx [@gu2006]. nAChR are also expressed in the postsynapatic neurons of the honeybee mushroom bodies based on the antibody staining against the nAChR as well as staining with labelled $\alpha$-Bgtx [@kreissl1989]. In addition, mushroom bodies produced a nAChR-like current in response to nicotine and ACh, and it was blocked by $\alpha$-Bgtx [@oleskevich1999]. In cocroach, cell bodies in the thoracic ganglion depolarised in the presence of nicotine and was blocked by selective antagonist mecamylamine [@bai1992].
<!-- Electrophysiology recordings from muschroom bodies - stimulation of antenal lobe - depolarisaion. In the presence of nAChR antagonist alpha-bgtx - synaptic response was inhibited [@oleskevich1999]. -->
<!-- TTx - tetrodoxin is a sodium channel blocker -in the presence of TTX no depolarisation in the post-ysynaptic neuron. -->
Generally, insects express ~10 nAChR subunits. *Apis mellifera* (Am) possesses 11 nAChR subunits: Am$\alpha1-9$, $\beta1-2$ [@jones2006], 8 subunits in *Mysuz Persicae* and there are 10 nAChR subunits in the Fruit fly *Drosophila melanogaster* [@jones2010]. The stoichiometry of these receptors in unknown, however there are some potential assemblies: *Drosophila* receptors containing $\alpha4\beta3$, $\alpha1\alpha2\beta2$ and $\beta1\beta2$ subunits [@chamaon2002]. This is based on the immunoprecipitation using antibodies against various receptor subunit types. Using a similar approach, it was shown that Nilaparvata lugens $\alpha1$, $\alpha2$ and $\beta1$, as well as $\alpha3$, $\alpha8$ and $\beta1$ co-assembly.
Heterologous expression of subunits in Xenopus oocytes hinted at the ehistance of other assemblies. Locust *Schistocerca gregaria* $\alpha1$ ($L\alpha1$) expressed in *Xenopus oocytes*, produced low current (low Nano amp values) with nAChR-like pharmacological and electrophysiological characteristics [@marshall1990]. *Myzus Persicae* $\alpha$ 1 and 2 [@sgard1998], were also successfully expressed. However, as in the case of Locust $\alpha1$, the expression was inconsistent and the currents produced by these channels in response to classical nAChR compounds were of low amplitudes (below 10 nA).
A number of insect subunits have been co-expressed with mammalian ones. When expressed alone, or in combination Drosophila $\alpha1$ or 2 as well as Nilaparvata lugens (Nl) $\alpha1$, 2 or 8 do not produce a functional receptor. However, a nAChR-like currents was produced in response to nAChR agonists upon expression of one of these subunits with rat $\beta2$ [@bertrand1994; @lansdell1997; @liu2009; @yixi2009]. A functional receptor was also obtained when Drosophila $\alpha2$ was co-expressed with chicken $\beta2$ [@matsuda1998] and when the three subunits: Nl $\alpha3$, $\alpha8$ and rat $\beta2$ were co-expressed [@yixi2009].
Taken together, there are many possible receptor assemblies in insects, however their stechiometry is largely unknown due to the diffuculties in expression these proteins in heterologous systems.
<!-- https://www.sciencedirect.com/science/article/pii/S0165614700018204#FIG2 - look at the cholinergic pathway -->
#### Mammals
Acetylcholine is a minor neurotransmitter in the vertebrate brain, acting to stimulate the release of other neurotransmitters [@kenny2000; @araujo1988]. nAChRs are expressed in the central nervous system and at the neuromuscular junction (NMJ) [@mcgehee1995] where they regulate cell excitability and muscle contraction, respectively. nAChRs are present in mechanosensory cells [@elgoyhen1994; @elgoyhen2001] and in the peripheral nervous system [@mcgehee1995]. In the CNS, nAChRs are situated on the pre-synaptic and post-synaptic membranes [@araujo1988; @fabian-fine2001] whereas at other locations, they are post-synaptic.
So far, 34 different vertebrate subunits have been identified [@millar2009] 17 of which are present in mammals. Mammalian subunits known to date are: $\alpha1-10$, $\beta1-4$, $\delta$, $\gamma$ and $\epsilon$ [@millar2009].
Much of the knowledge of the stechiometry of the muscle type receptor is derived from work involving the Torpedo Fish, muscle-type nAChR. The anatomy of Torpedo and mammalian muscle type receptors is similar, therefore the former served as a platform to study vertebrate subunit stechiometry.
In 1985, mRNA encoding for 4 receptor subunits, namely $\alpha$, $\beta$, $\delta$ and $\gamma$ were injected into the *Xenopus oocytes* [@mishina1984]. Upon application of ACh, a current was recorded, suggesting a functional expression. Receptor was also formed from three subunits lacking $\delta$, however responses to agonists were reduced. In the absence of any other one of the subunits, the responses to ACh were either absent or greatly reduced, therefore 4 subunits are required for the normal function of this protein.
<!-- Function of this receptor studied by voltage clamp - either ACh responses or I-bgtx binding -->
@claudio1987 expressed the same receptor but instead of using *Xenopus oocytes*, he used cultured mammalian cell lines. Ligand binding studies with $\alpha$-Bgtx and electrophysiological recordings upon acetylcholine application confirmed functionality of this receptors.
The structure of Torpedo receptor was established by electron microscopy of deep frozen receptors embedded in the post-synaptic membrane and revealed a pentameric arrangement of $\alpha1-\beta1-\delta-\alpha1-\gamma$ subunits, in the clockwise order [@unwin1993]. The variation in the vertebrate receptor stechiometry at different stages of the development was shown, using bovine receptor; $\gamma$ subunit in present in the embryo, but becomes replaced by the $\epsilon$ subunit in adults [@mishina1986]. These receptors have different pharmacological properties, demonstrating that the receptor anatomy can have a dramatic impact on the receptor pharmacology.
Combined mutagenesis, heterologous expression in *Xenopus oocytes* and electrophysiological studies revealed stechiometry of receptors expressed in the vertebrate brain [@cooper1991]. $\alpha4$ and $\beta2$ injected into the oocytes generated functional receptor. Reporter mutations in both subunits led to dramatic change in conductance, which was distinct from the conductance exhibited by receptors with only 1 mutated subunit. Based on these observations, it was concluded that two $\alpha4$ and three $\beta2$ subunits form a receptor.
Using a similar approach stechiometry of $\alpha3\beta4$ was established in which 2 $\alpha$s and three $\beta$s are present [@boorman2000].
The stechiometry of muscle and $\alpha4\beta2$ type receptors was confirmed by the biochemical method [@anand1991]. However, alternative assemblies of these receptors may exist. For example, $\alpha4\beta2$ in 2:3 ratio [@nelson2003]. This receptor has distinct pharmacological properties, suggesting that not only the identity of subunits but also the subunit ratio effects pharmacological profile of nAChRs.
<!-- . Relative amount of the amount of label in radiolabelled receptor assemblies were calculated. The ration between alpha and beta subunit was 1:1.5. Since nAChR is a pentamer, the a4b2 subunit is in the $\alpha4$~2~$\beta2$~3~ . Similar approach was applied to confirm stechiometry of muscle and $\alpha3\beta4$ receptor. -->
Most vertebrate receptors are formed from a combination of $\alpha$ and $\beta$ subunits [@wang1996a; @anand1991], however there are some that preferentially form homopentamers. One such receptor is $alpha7$.
$\alpha7$ is a neuronal type [@keyser1993] receptor which forms homooligomeric receptors. The homomeric nature of this receptor was confirmed a diversity of methods. Proteins binding to nAChR were isolated from rat membrane fractions with $\alpha$-Bgtx affinity column. A single protein species was isolated which exhibited with high binding affinity to $\alpha7$ compounds and were stained by $\alpha7$ antibodies [@chen1997; @drisdel2000]. In additional electrophysiological recordings and radiolabelled studies confirmed that upon injection of $\alpha7$ cDNA into *Xenopus* oocyte [@couturier1990] or mammalian cell lines [@cooper1997; @gu2016], a functional receptor is formed.
$alpha7$ can assemble with other subunits. Injection of rat $\alpha7$ and $\beta2$ subunits into Xenopus oocytes resulted in formation of a channel with distinct pharmacological and kinetic properties than those of homomeric $\alpha7$ [@khiroug2002]. Co assembly of $\beta3$ with $\alpha7$ was shown by the binding of radiolabelled $\alpha$-Bgtx, however no functional response to acetylcholine was recorded [@palma1999].
$\alpha7$ homopentamers and $\alpha4$$\beta2$ heteropentamers are the most abundant nAChRs in the vertebrate brain [@tomizawa2001]. There are also $\alpha8$ [@gerzanich1994] and $\alpha9$ [@elgoyhen1994] homopentamers and many other heteropentameric receptors [@millar2009].
<!-- This paper also described responses to nicotine, bgtx and ach. Same paper Co-injection with other subunits - i.e. alpha1, 2, 3 and c-injection with 3 subunits: delta, gamma and beta - receptor with the same current properties, suggesting it does not co-assemly -->
<!-- Also succesful expression of rat alpha7 in cultured mammalian cell lines [@cooper1997] I-bgtx labelling here -->
<!-- Alpha7 monoclonal antibodies also here . Note that this paper purifies proteins from pheochromocytoma 12 (PC12) with . And purifies only alpha7 (despite other subunits potentially being there, as mRNA for $\alpha3,\alpha5, \beta2 and \beta4$ are being detected in these cells [@chen1997]. -->
<!-- [@palma1999] Some characteristics and pharma described here i.e. ACh -->
<!-- . Note that this paper dewxribes kinetics quite nicely and pharma in response to ACh, carbachol and choline -->
<!-- Biochemical evidence - mammalian cell lines transfected with beta2 and epitope tagged alpha7. Cells incubatesd with radiolabelled [35S]methionine and [35S]cysteine and examined by immunoprecicitation. Cell lysates were immunoprecipitated with beta2 specific monoclonal ab. Co-precicitation of alpha7 FLAG by the monoclonal anti beta2 ab was seen when the two subunits were expressed together -->
<!-- Effects on insect receptors - activation of receptors at low doses, inhibition, paralysis and death at higher doses -->
#### Nematode {#nematodenachrs}
Acetylcholine is the major neurotransmitter in the nervous system of nematodes. nAChRs are expressed at the neuromuscular junction [@mcgehee1995] and in the nervous system [@lewis1987]. The representative of phylum Nematoda and a model organism *C. elegans* contains 29 genes encoding for nAChR subunits [@jones2007b], 22 of which are $\alpha$-subunits (Table \@ref(tab:celegans-nachrs)). The receptor subunits are assigned to five groups based on the sequence homology: DEG-3, ACR-16, ACR-8, UNC-38, and UNC-26.
So far, only a few functional nAChRs have been identified. Neuronal DES-2/DEG-3 form a functional receptor in *Xenopus* oocytes [@treinin1998]. There are two receptor at the body wall muscle differentiated by those more sensitive to anthelmintic levamisole (L-type) and those more sensitive to nicotine (N-type) [@richmond1999]. The subunit composition of these receptors is respectively: UNC-29, UNC-38, UNC-63, LEV-1, LEV-8 associated with auxiliary subunits RIC-3, UNC-50, and UNC-74 [@boulin2008] and ACR-16 homopentamer [@touroutine2005]. EAT-2 is a predicted $\beta$ nAChR expressed in the pharyngeal muscle, believed to assemble with auxilary subunit EAT-18, based on common localisation and behavioural phenotypes of *eat-2* and *eat-18* mutants [@mckay2004]. ACR-2 and UNC-38 may also co-assembly; co-injection of cDNAs encoding for both subunits into *Xenopus*, produced currents in response to high concentration of levamisole [@squire1995]. These currents were of low amplitude, suggesting auxilary proteins are required for a normal function of this receptor.
```{r celegans-nachrs, echo=FALSE, message = FALSE, warning=FALSE}
library(kableExtra)
library(dplyr)
celegans_nachrs <- data.frame(
Group = c("DEG-3", "ACR-16", "ACR-8", "UNC-38", "UNC-29"),
Subunits = c("ACR-17, ACR-18, ACR-20, ACR-22*, ACR-23, DES-2, DEG-3\nACR-24, ACR-5", "ACR-7, ACR-9*, ACR-10, ACR-11, ACR-14*, ACR-15\nACR-16, ACR-19, ACR-21, ACR-25*, EAT-2*", "ACR-8, ACR-12, LEV-8", "UNC-38, UNC-63, ACR-6", "ACR-2*, ACR-3*, UNC-29*, LEV-1*"))
celegans_nachrs %>%
mutate_all(linebreak) %>%
kable(format = "latex", align = "l", booktabs = TRUE, escape = FALSE,
caption = 'nAChR subunits in \\textit{C. elegans}.') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
footnote(general= " Non-alpha subunits are marked with *. Figure modified from Holden-Dye el al., 2013.",
threeparttable = T)
```
### Pharmacology
Such diversity in receptor subunits and subunit assemblies leads to differences in pharmacological profiles of nAChR and underpins selectivity of several pharmacological agents to certain receptor proteins.
<!-- These neuronal effects are reflected in the behavioural data [@sone1994]. Upon exposure to toxic dose of neonicotinoids, insects udergo convulsions, uncoordinated movement, tremors as well as feeding inhibition, eventual paralysis and death [@suchail2001; @boiteau1997; @alexander2007]. -->
#### Neonicotinoids
Neonicotinoids bind to high affinity to insect nAChRs, in comparison to human or nematode receptors. In cultured honeybee mushroom bodies, imidacloprid acts at nM concentrations [@palmer2013]. In the bee Kenyon cells, the EC50 of imidacloprid was 25 $\mu$M [@deglise2002], whereas on the antenna lobe cells 0.87 $\mu$M [@nauen2001]. Similarly, the EC50 of imidacloprid on isolated cockroach neurons [@tan2007; @ihara2006] and in Drosophila CNS neurons [@brown2006] were in a single digit $\mu$M range. Neonicotinoids are less potent on vertebrate receptors. EC50 of clothianidin and amidacloprid on heterologously expressed human $\alpha7$ nAChRs is 0.74 mM and 0.73 mM, respectively [@cartereau2018]. Whereas the EC50 of imidacloprid on heterogouslty expressed chicken $\alpha7$ is 357 $\mu$M [@ihara2003]. Other vertebral receptors may be more susceptible. Two-digit $\mu$M imidacloprid doses were needed to activate nAChRs in mammalian neurons [@bal2010; @nagata1998], and the EC50 of this compound on cells containing native mammalian $alpha4\beta2$ nAChRs is 70 $\mu$M [@tomizawa2000a]. Higher EC50 of neonicotinoids on vertebrate receptors suggests neonicotinoids bind preferentially to the insect nAChR. This supports the toxicological data demonstrating increased toxicity of neonicotinoids on insects versus vertebrate species (Section \@ref(nontargeteffect)).
<!-- No pharmacological data on human alpha7, but radiolabelled ligand studies in which the afinity of rat brain homogenate to IMI-analogue was tested showed low affinity [@kagabu2000]. -->
#### Acetylcholine
Acetylcholine is an endogenous nAChR agonist expressing similar affinities to all nAChRs. Based on the receptors expressed in Xenopus oocytes, the EC50 on mammalian $\alpha7$ is 21 $\mu$M [@papke2002], whereas on N- and L-type *C. elegans* receptors, it is 31 and 26 $\mu$M, respectively [@touroutine2005; @boulin2008]. EC50 on insect chimera receptors varies between ~10 and ~60 $\mu$M. It is 17.6 $\mu$ at Nl $\alpha$ 3, $\alpha8$ and rat $\beta2$, [@yixi2009], 15 $\mu$M at Drosophila $\alpha2$ chicken $\beta2$ [@lansdell1997] and 63 $\mu$M on the Locusta migratoria manilensis $\alpha1$/rat $\beta2$ [@bao2015].
#### Choline
Choline is a breakdown product of acetylcholine at the synapse [@fukuto1990]. Its affinity to nAChRs is much lower; the EC50 on human $\alpha7$ receptors expressed in Xenopus oocytes is 14 $\mu$M [@papke2002]. $\alpha7$ seems to be the most choline-sensitive vertebrate nAChR.
#### Nicotine
Nicotine is an exhogenous agonist, naturally occuring in Tabacco plant. It is a potent activator of some insect nAChRs. Its EC50 was 0.88 $\mu$M on cultured cells of *Apis mellifera* [@wuestenberg2004] and 1 $\mu$M on isolated *Drosophila* neurons [@wegener2004]. The EC50 of 100 $\mu$M was noted on heterologosuly expressed Locusta migratoria manilensis $\alpha1$/rat $\beta2$ receptors [@bao2015]. Thus it is possible that some insect receptors exhibit low sensitivity to nicotine.
In *C. elegans*, the potency varies between receptors. At 100 $\mu$M, nicotine had no effects on L-type receptors [@boulin2008], but it activated N-type receptors in a dose-depenent manner and with the EC50 of 12.6 $\mu$M [@ballivet1996]. 12.6 $\mu$M is also an EC50 against the human $\alpha7$ receptors [@ballivet1996].
Taken together, it seems that nicotine acts on insect, human and worm nAChRs. However, within each species, there might be receptors more and less sensitive to this agonist.
#### Levamisole
Levamisole is a synthetic compound used in treatment of parasitic worm infestation in both humans and animals [@miller1980]. It has high potency against worm receptors. In *C. elegans*, it is an agonist at L-type nAChRs with the EC50 of 10.1 $\mu$M [@boulin2008] and an antagonist of N-type receptors with the IC50 of 36.0 $\mu$M [@ballivet1996]. Levamisole also activates Drosophila nAChRs with low affinity [@pinnock1988] but has no effect on mammalian $\alpha7$-chimera receptors [@bartos2006].
#### Cytisine
Cytisine is a naturally occurring compounds found in *Fabaceae* family of plants. In mammals, it is selective for $\alpha7$ nAChRs which display an EC50 value of 5.6-7.1 $\mu$M [@papke2002]. Cytisine is a partial agonist on Apis mellifiera nAChRs with much lower effective doses, in comparison to the human $\alpha7$. Electrophysiological recordings from the cultured Kenyon cells show weak current in response to 3 mM and no response to 0.5 mM [@wuestenberg2004].
##### $\alpha$-Bgtx
There is a great diversity of pharmacological agents activating nAChRs but there are also compounds which block these proteins.
$\alpha$-bungarotoxin is a snake venom neurotoxin. It is a 74- amino acid long and 8 kDa protein that acts as a competitive nAChR antagonist [@mishina1984]. At 100 nM, $\alpha$-bungarotoxin inhibits acetylcholine-induced $\alpha7$ receptor current in Xenopus oocytes [@couturier1990]. At 1 $\mu$M, it also inhibits ACh-evoked current in the honeybee Kenyon cells [@wuestenberg2004] and at nM concentrations inhibits nAChR in cultured Drosophila and Musca (fruit and domestic fly, respectively) @albert1993]. $\alpha$-bgtx does not block nematode nAChRs. It had little antagonistic effect on the N- or L- type *C. elegans* receptors or N-type receptors of the parasitic nematode Ascaris suum expressed in Xenopus oocytes [@ballivet1996; @boulin2008; @abongwa2016].
\newpage
```{r echo=FALSE, message = FALSE, warning=FALSE}
library(kableExtra)
library(dplyr)
pharma_overall <- data.frame(
Compound = c("Imidaclorid", "Clothianidin", "Acetylcholine", "Acetylcholine", "Acetylcholine", "Acetylcholine", "Nicotine", "Nicotine", "Nicotine", "Nicotine", "Nicotine", "Levamisole", "Levamisole", "Levamisole", "Cytisine", "Cytisine"),
Species = c("Honey bee", "Human", "Planthopper/Rat", "Human", "C. elegans", "C. elegans", "Honey bee", "Locust/Rat", "Human", "C.elegans", "C. elegans", "Cocroach", "Human", "C. elegans", "Honey bee", "Human"),
Receptor = c("Unknown", "$\\alpha7$", "$\\alpha3\\alpha8\\beta2$", "$\\alpha7$", "L-type", "N-type", "Unknown", "$\\alpha1\\beta2$", "$\\alpha7$", "L-Type", "N-type", "Unknown", "$\\alpha7$5HT", "L-type", "Unknown", "$\\alpha7$"),
Source = c("Kenyon cells", "Xenopus oocytes", "Xenopus oocytes", "Xenopus oocytes", "Xenopus oocytes", "Xenopus oocytes", "Kenyon cells", "Xenous oocytes", "Xenopus oocytes", "Xenopus oocytes", "Xenopus oocytes", "Motor neuron", "Xenopus oocytes", "Xenopus oocytes", "Kenyon cells", "Xenopus oocytes"),
EC50 = c("25 $\\mu$M", "0.75 mM", "17.6 $\\mu$M", "21 $\\mu$M", "26 $\\mu$M", "31 $\\mu$M", "0.88 $\\mu$M", "12.6 $\\mu$M", "100 $\\mu$M", ">100 $\\mu$M", "12.6 $\\mu$M", " ", ">1 mM", "*36 $\\mu$M", "> 3mM", "5.6-7.1 $\\mu$M"),
Reference = c("Deglise et al., 2002", "Cartereau et al., 2018", "Yixi et al., 2009", "Papke et al., 2002", "Boulin et al., 2008", "Touroutine et al., 2005", "Wuestenberg et al., 2004", "Bao et al., 2015", "Ballivet et al., 1996", "Boulin et al., 2008", "Ballivet et al., 1996", "Ballivet et al., 1996", "Pinnock et al., 1988", "Bartos et al., 2006", "Wuestenberg et al., 2004", "Papke et al., 2002"))
pharma_overall %>%
kable("latex", align = "l", escape = F, booktabs = TRUE,
caption = "Pharmacology of insect, human and nematode nAChRs",) %>%
kable_styling (position = "center", full_width = FALSE, latex_options = c( "hold_position", "scale_down")) %>%
footnote(general= " ",
threeparttable = T)
```
## Neonicotinoids act by targeting nAChRs
Effects of neonicotinoids on insects are mainly derived from voltage clamp recordings from the neurons expressing nAChRs, whereby in response to the application of neonicotinoids and related compounds, a depolarisation followed by a neurotransmission block of the post-synaptic membrane at the cholinergic synapses of insects was observed.
<!-- In electrophysiological studies, it was shown that neonicotinoid depolarises post-synaptic cholinergic neurons of the isolated nervous system of Colorado potato beetle and/or cocroach [@buckingham1997; @thany2009; @sattelle1989; @tan2008]. -->
<!-- Imidacloprid also depolarises the the cell body of motor neurons of the cocroach [@sattelle1989]. -->
Repetitive firing of neurons in response to neonicotinoids followed by a block of neuronal activity from the isolated thoracic ganglia of male adult American cockroaches, *Periplaneta americana* [@sone1994; @tan2007; @thany2009; @ihara2006] and the Colorado potato beetle, *Leptinotarsa decemlineata* [@tan2008] was recorded. In Kenyon cells of the honeybee brain, a depolarisation of the post-synaptic neuron was seen [@palmer2013]. These neonicotinoid-induced effects were prevented by application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine) [@buckingham1997; @thany2009; @tan2008], suggesting neonicotinoid-induced currents are due to the activation of nicotinic and not muscarinic receptors.@crespin2016 extracted membrane preparations of pea aphid *Acyrthosiphon pisum* and injected them into the Xenopus oocyte. Upon application of classical agonists of nAChRs acetylcholine and nicotine as well as neonicotinoid clothianidin, the nAChR-like current was evoked. In addition, using both brain homogenate and isolated hybrid nAChRs it was shown that the binding affinity of neonicotinoids and their agonist activity correlates well with their insecticidal activity [@nishiwaki2003], in agreement with studies on the domestic fly [@kagabu2002] and brown planthopper [@liu2005].
Resistance of insects to neonicotinoids arises from the mutation in nAChRs. A strain of Myzus persicae developed resistance to imidacloprid due to the mutation in nAChR subunit [@bass2011].
## Mode of action
Neonicotinoids can have diverse mode of action, with some acting as partial-, true-, super-agonist or antagonists. The current 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 are true agonists, and those more efficacious than ACh are 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 diverse mode of action on different preparation implies there are distinct nAChRs in American cocroach.
Clothianidin acts as a super agonist on nAChRs expressed in culture Drosophila CNS cholinergic neurons, whereas imidacloprid is a partial agonist there [@brown2006]. Clothianidin and imidacloprid are partial and full, agonist, respectively on the isolated Kenyon cells of the honeybee [@palmer2013]. In addition imidacloprid, and imidacloprid but not other neonicotinoids tested blocked ACh-induced action on the American cockroach neurons [@ihara2006] and isolated honeybee neurons [@palmer2013], suggesting antagonistic capabilities of these compounds. Single channel recordings provided a mechanistic details of the mode of action of these compounds. Super-agonists have been shown to increased the frequency of larger conductance state openings [@jones2007a], whereas partial agonists increase the activity of the sub- conductance state of nAChRs [@nagata1996; @nagata1998].
## Structure of nAChRs
The majority of existent knowledge regarding the shape and size of the nAChR originates from the structural data of the muscle type nAChR from its rich source- electric organ of electric ray Torpedo, obtained utilising cryo- and electron-microscopy (Figure 1.2a and 1.2b). The receptor protein is in the shape of an elongated, 125 Å funnel [@unwin1993; @toyoshima1990]. It consists of large, extending to the synaptic space [@toyoshima1990] N-terminal ligand binding domain [@sigel1992], the membrane spanning pore-domain [@eisele1993], intracellular MA helix [@toyoshima1990; @unwin1993], and C-terminus positioned extracellularly (Figure 1.2a). Constituting nAChR subunits are arranged pseudosymmetrically, around the central ion conduction pore [@brisson1985]. The subunit composition of the neuromuscular nAChR follows the strict order of $\alpha1$, $\beta1$, $\delta$, $\alpha1$, $\gamma$ (clockwise). Each subunit of the nAChR contains 4 transmembrane helices [@noda1982; @noda1983] named M1, M2, M3 and M4, as moving from N- to C- terminus. M1, M3 and M4 are exposed to the plasma membrane [@blanton1994], shielding M2, pore-forming helices [@imoto1986; @hucho1986] from the hydrophobic environment of the bilayer. As the outer helices progress from the outer to the inner leaflet of the membrane, they tilt inwards [@miyazawa2003], narrowing down the width of the channel. M2 on the other hand, bends roughly in the middle of the bilayer [@unwin1995], where it forms the most restricted part of the ion conductivity pathway. There are hydrophobic interactions between the outer helices, which stabilise the outer wall of the receptor and hence limit the conformational changes adopted by the inner helix. In contrast there are no extensive bonds between the inner and outer helices [@miyazawa2003]. As lining pore structures, the inner helix and flanking sequences contain molecular determinants for ion selectivity, permeability, the rate of conductance and gating. These were investigated by pharmacological, biochemical and electrophysiological approaches. [@imoto1988; @imoto1991; @konno1991] investigated the function of several rings of anionic and neutral amino acids with side chains facing towards each other in the centre of the pore. The so called intermediate ring (constituting of αE241 and equivalent) and the adjacent to $\alpha$ E241 in helical configuration central ring, (formed by $\alpha$ L244 and equivalent) form a narrow constriction of the ion pore, hence have the strongest effect on the conductance rate [@imoto1991; @imoto1988]. In addition, the negatively charged side chains of intermediate ring are crucial for ion selectivity [@konno1991]. The gating of the channel is governed by conserved leucine residues, slightly towards the extracellular side from the centre of the bilayer with side chains projecting inwards [@unwin1995], hence occluding the passage for ions.
ALSO TALK ABOUT THE NEGATIVELY CHARGED VESTIBULE HERE
(ref:structure-nachr) **Structural features of the nicotinic acetylcholine receptor.** Torpedo nAChR is a transmembrane protein, made up of 5 subunits (colour-coded), arranged around the ion conductivity pore. Each subunit consists of extracellular ligand-binding, transmembrane and intracellular domain (a) (PBD code:2BG9). Extracellular domain of a single subunit consists of 10 $\beta$-strands and N-termical $\alpha$-helix. It contains a disulphide bridge between Cys192 and Cys193 (highlighted in yellow) (b). Fully formed receptors have five ligand binding pockets formed by the contributions from the neighbouring subunits (A-B, B-C, C-D, D-E and E-A), named the principle and the adjecent components, respectively. Top view of the molluscan AChBP (PDB:1I9B) with amino acids forming the agonist binding site in ball and stick representaion (c). Images generated with the UCSF Chimera software.
```{r structure-nachr-label, fig.cap="(ref:structure-nachr)", fig.scap='Structural features of the nicotinic acetylcholine receptor.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/crystal_structure_nachr.png")
```
Determination of the crystal structure of the molluscan acetylcholine binding protein [@brejc2001] (Figure 1.2c) provided a platform to study the ligand binding domain of nAChRs. Acetylcholine binding protein (AChBP) is a soluble protein, secreted by snail glial cells into the cholinergic synapses to bind released ACh and modulate neurotransmission [@sixma2003]. It shares 24 % sequence identity with mammalian $\alpha7$ homopentameric receptor. It is has similar structure to the extracellular domain of the nAChRs mammalian alpha1 [@dellisanti2007] and $alpha7$ [@li2011]. It is a homopentamer with N-terminal helix and 10 $\beta$sheets. It also shares similar pharmacological properties to this receptor. AChBP binds to classical nAChR agonist and antagonists: nicotine, acetylcholine and $\alpha$-bungarotoxin [@smit2001]. Therefore AChBP is considered a good model for the nAChR ligand-binding domain structural studies. The structures of AChBP inactive [@brejc2001], bound to agonist and antagonist [@celie2004; @hansen2005], chimera $\alpha1$ [@dellisanti2007] and $\alpha7$ are known [@li2011]. The common structural features of the ligand binding site emerge from all available data. Here data from Ls will be discussed (it unlike Ac, all aromatic residues in Ls are conserved).
### Binding site
The nicotinic acetylcholine receptor binding pocket is formed on the interface of the adjacent subunits [@brejc2001; @middleton1991; @blount1989] (Figure \@ref(fig:binding-pocket-label)). In case of the neuromuscular heteropentameric receptors, it constitutes of $\alpha$ and non-$\alpha$ subunit contributions, whereas in homopentameric or $\alpha$ heteropenameric receptors it is made up of neighbouring subunits. The principal, $\alpha$-subunit site subsidises amino acid side chains originating from discontinuous loops A (loop $\beta4$-$\beta5$), B (loop $\beta7$-$\beta8$) and C (loop $\beta9$-$\beta10$), whereas the complementary (non-$\alpha$) subunit contributes amino acid side chains originated from loop D (loop $\beta2$-$\beta3$), E (loop $\beta5$-$\beta6$) and F (loop $\beta8$-$\beta9$). Specific residues involved in the formation of the ligand binding pocket were depicted by the molluscan AChBP (Figure \@ref(fig:binding-pocket-label)). Amino acids of the principal component are: Tyr93, Trp147, Tyr188 and Tyr195, whereas non-$\alpha$ component contributes Trp53, Gln55, Arg104, Val106, Leu112 and Met114, Tyr164.
(ref:binding-pocket) **The ligand binding domain of acetylcholine binding protein.** Agonist binds to the loops situated in the adjecent subunits of the nAChR. In muscle type receptor, there are 2 binding sites, and thee are 5 in homopentameric receptor, such as mammalian $\alpha7$ (a). The ligand binding pocket of the AChBP (PDB:1I9B) is formed from loops of the neighbouring subunit (b). Principal and complementary subunits contributed amino acids from loops A, B, C and D, E, F, respectively (c). Crystal structure of the AChBP generated with the USCF Chimera software.
```{r binding-pocket-label, fig.cap="(ref:binding-pocket)", fig.scap='The ligand binding domain of acetylcholine binding protein.', fig.align='center', echo = FALSE}
knitr::include_graphics("fig/general_intro/png/binding_pocket_3.png")
```
### Pharmacophore
<!-- How do diverse structural compounds bind to nAChR? ACh and nicotine as well as their analogoues typically have two features : bond acceptr and cation donor. Bond acceptor is either oxygen of the carbonyl group, or nitrogen -->
<!-- Acetyl and quaternary ammonium. Carbonyl oxygen of the acetyl group is a bond acceptor. -->
<!-- Nitrogen from the quaterny ammonium is protonated at physiological pH. -->
<!-- Nicotine pyridine (6-membered ring) and pyrrolidine (5-memebred ring). Pyridine contains -->
The pharmacophore of agonists: cationic nitrogen and a bond acceptor. Cationic nitrogen is tertiary amine in nicotine and quaternary amine in ACh are protonated at physiological pH. Bond acceptor is more diverse and oxygen of carbonyl or nitrogen of pyridine ring.
Crystal structure of the AChBP bound to ACh anatogue carbamylcholine, nicotine [@celie2004] and its analogue epibatidine [@hansen2005] provided some general features of the binding pocket. More recently, structures of mammalian receptors: $\alpha9$ [@zouridakis2014] bound to methyllycaconitine, $\alpha2$ extracellular domain bound to epibatidine [@kouvatsos2016] and $\alpha4\beta2$ receptor bound to nicotine [@morales-perez2016] have been obtained. These, together with the AChBP structures provide details of how structurally varied agonists bind to the nAChRs. Agonist are buried on the interface of the neighbourig subunits. They are stabilised in the binding pocket 5 conserved aromatic residues from A, B and C loops of the principal site (known as the aromatic box) engulf the cationic nitrogen of bound agonist. There are two major and conserved features: caption - pi intreaction and hydrogen bond.
Cation -$\pi$ interactions are formed between the cationic nitrogen and aromating side chain of tryptophan in loop B (143 in AChBP) of the principal side of the binding pocket. Whereas hydrogen bond is formed between the bond acceptor and amino acids of the complementary side of the binding pocket via water molecule [@celie2004; @olsen2014]. In ACh and nicotine bound to AChBP water bridges to the oxygen of the carbonyl group of Leu112 and amide group of Met114 in loop E [@olsen2014; @celie2004].
Choline is an agonist lacking the hydrogen bond acceptor, which is likely contributing to its low binding affinity. So for example, on human alpha7 receptor, the EC50 of choline = 0.4 - 1.6 mM, whereas nicotine 49 - 113 $\mu$M [@wonnacott2007]. And binding affinity in radiolabelled studies of ~ 2mM in comparison to nicotine 0.4 - 15 $\mu4$M [@wonnacott2007].
There are also some less conserved features between agonists and the principal side of the binding pocket. For example, in AChBP-nicotine structures, there is a hydrogen bond between cationic nitrogen and carbonyl of TrpB [@celie2004]. Similarly, in human $\alpha2$ structures a hydrogen bond between the cationic nitrogen of apibatidine and carbonyl of TrpB or Tyr in loopA is formed [@kouvatsos2016]. In contrast, cationinc nitrogen of ACh forms cation-pi with Trp53 in loop D of AChBP and $\alpha2\beta2$ proteins [@morales-perez2016; @olsen2014].
(ref:pharmacophore) **Nicotinic acetylcholine receptor agonist pharmacophore.** Agonists of the nAChRs contain hydrogen bond acceptor (red) and cationic nitrogen (blue). Interactions with the receptor based on the crystal structure of the AChBP and nicotine (PB:1UW6) b. Chemical structures generated in ChemDraw. b taken from @blum2010. Cation-pi interactions between protonated nitrogen of tertriaty amine and indole of TrpB.
```{r pharmacophore-label, fig.cap="(ref:pharmacophore)", fig.scap='Nicotinic acetylcholine receptor agonist pharmacophore.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/nicotinic_interactions.png")
```
### Neonicotinoid-pharmacophore
<!-- Radiolabelled, electrophysiological and structural studies revealed that neonicotinoids bind to the orthostatic ligand binding site of the nAChR, and that they are selectively active on insect nAChRs (Matsuda et al., 2001). -->
One of the major differences is that at physiological pH, neonicotinoids are predominantly deprotonated, whereas nicotine is predominantly protonated. Therefore nicotine containing cationic nitrogen is electropositive, whereas cyano or nitro-group of neonicotinoids has electronegative tip [@tomizawa2003].
Structure of AChBP proved to be valuable in determining structural elements which may account for neonicotinoids’ selectivity @ihara2008; @talley2008 derived crystal structures of the great pond snail (Lymnaea stagnalis, Ls) and California sea slug (Aplysia californica, Ac) AChBP complexed with bound neonicotinoids (imidacloprid, clothianidin, thiacloprid), and non-selective nAChR ligands- nicotinoids (nicotine, epibatidine and desmotroimidacloprid). Comparison of these structures revealed differences in binding modes between nicotinoids and neonicotinoids described below (see Appendix \@ref(fig:pharacophore-seq-label) for sequence alignment), which allowed for predictions of the binding interactions between neonicotinoids and insect receptors (Figure @\ref(fig:)) The positioning of the pyridine ring of neonicotinoids and nicotinoids is virtually identical. In imidacloprid bound structure, nitrogen forms hydrogen bond with the amide group of Met114 and carbonyl group of Leu102 of loop E, via water molecule. The presence of water molecule, position of the nitrogen and similar interactions are also seen in nicotinic-bound structures [@celie2004; @ihara2008; @talley2008). In addition, chlorine atom of imidacloprid, thiacloprid as well as epibatidine makes van der Waals interactions with oxygen of Ile106 and oxygen of Met116 of AChBP.
One of the main features of the nicotinic is the presence of the cation-pi interations between the cationic nitrogen of the pyrrolidine ring and TrpB. In imidacloprid bound structures, the ring stacks with aromatic residues Tyr185 and Tyr188 of loop C. These stacking interactions result in the formation of CH-pi intreactions between the methyline bridge (CH2-CH2) of imidacloprid and TrpB.
The 5-membered ring of imidacloprid stacks with evolutionary conserved tyrosine residue (Tyr188 of Ac and Tyr 185 of Ls), which is also seen in epidibine, but not in nicotine. All residues described so far are conserved in nAChRs, therefore do not account for neonicotinoids-selectivity.
The differences come to light when one begins to dissect the interactions between imidacloprid ring substituents and the AChBP. Partially positive nitro group (NO2) bridges to glutamine of loop D (Gln55 in Ls and Glu 57 in As) via hydrogen bond. It is interesting that in some nAChR subunits, such as chicken $\alpha2$, honeybee $\alpha6$, $\alpha7$, $\beta1$ and $\beta2$, glutamine corresponds to basic residue (lysine/arginine). Basic residues electrostatically attract nitro group, possibly forming a hydrogen bond, which in turn would strengthen the stacking and aromatic CH/π hydrogen bond interactions between the ring and the protein. In contrast, other subunits contain either acidic or polar amino acids in the exact position, repulsing or forming no electrostatic interactions with imidacloprid. Thus it has been proposed that the basic residue in loop D interacting with the nitro group of the imidazole ring
(ref:imi-binding) **The predicted interactions between imidacloprid and insect nAChR.** Electrostatic interactions between the nitro group of imidazole ring of imidacloprid and basic residue of Loop D strengthens the interactions between neonicotinoids and nAChRs. Imidazole ring forms stacking interactins with Tryptophan of loop B. Its position is also stabilised by the Tyrosines of loops A and C. Carbonyl group of TrpB can form alternative interaction with imidaclopid. Image taken and modified from @matsuda2005.
```{r imi-binding-label, fig.cap="(ref:imi-binding)", fig.scap= "The predicted interactions between imidacloprid and insect nAChR", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/imi_binding_site.png")
```
Therefore this residues may be important in confirming neonicotinoid selectivity in insect nAChR subunits. This is supported by the genetic studies. Loop D arginine to threonine mutation naturally occurring in $\beta1$ subunit of peach aphid Myzus persicae, and coton aphid Aphis gossypii [@hirata2017; @bass2011; charaabi2018] gives rise to neonicotinoid resistance. Additionally, Shimomura et al showed that mutation of glutamine in loop D of human $\alpha7$ to basic residue, markedly increases sensitivity of the $\alpha7$ homopentamer to nitro-containing neonicotinoids [@shimomura2002], whereas mutation of loop D threonine to acidic residues in chicken $\alpha4\beta2$ and hybrid chicken/drosophila $\alpha2\beta2$ receptor had an opposite effect [@shimomura2006]. Interestingly, described mutations did not influence the efficacy to nicotinoids, suggesting this interaction is specific to neonicotinoids. Above evidence the residues on the
complementary site are more variable and they are important in determination of the neonicotinoid selectivity. This is in agreeement with other data.
This supports the ideas that selectivity is determined by the complemetary subunit amino acids [@marotta2014]. Swapping of $\beta$ subunits in heteromeric recepor diminished cytisine activity on the receptor [@harpsoe2013]. Receptor becomes
responsive to cytisine at sub-$\mu$M concentrations by a mutation in a single amino acid in the complementary binding pocket [@marotta2014]. The variation in this binding pocket residues gives rise to ligand binding specifities and pharmacological differences between various compounds and receptors. So for example, conserved Trp in loop D is essential for the high affinity binding of $\alpha$-Bgtx to Ac AChBP [@hansen2004].
<!-- Some subunits recognised as those potentialy sensitive to neonicotinoids, such as locust $\alpha1$, Myzus $\alpha1$ and $\alpha3$, Nl $\alpha3$ and Drosophila $\alpha1-3$, all contain glutamate. What is more, some subunits not-sensitive to nicotinoids such as Human $\alpha1-4$ contain lysine. OK but these subunits are alpha, whereas Gln55 is in beta subunit -->
This could mean that basic residue in this position is critical to confer neonicotinoid sensitivity in some, but not in all subunits, or/and that there are other structural determinants necessary to confer sensitivity to these drugs.
Note that the following proline is in the alpha subunit
Here, of particular interest are: residue X in the YXCC motif of the C loop, and basic residue in loop G of the nAChR. Studies on drosophila/chicken $\alpha2\beta2$ hybrid and chicken $\alpha2\beta4$ receptors showed that the presence of nonpolar proline in YXCC motif enhances affinity, whereas mutation of proline to glutamate markedly reduces affinity of neonicotinoids to these receptors [@shimomura2005]. This proline is in the loop C
Although both Ls and Ac AChBPs have serine at this position, they make different interactions: LsAChBP serine makes interactions with Glu163 of loop F, whereas in AsAChBP serine is hydrogen bonded to nitric group of imidacloprid. The former seems to be more likely duplicated in nAChRs, as based on homology model of $\alpha4\beta2$ bound to imidacloprid. Following this thought, the presence of acidic amino acid in loop C would lead to electrostatic repulsion with acidic residue in loop F correspondent to Glu163 in LsAChBP. Based on the primary amino acid sequence, such interaction is possibly present in chicken β2 and β4. On the other hand, locust $\alpha1$, cat flee $\alpha3$ and fruit fly $\alpha2$ all have proline in this position which could contribute to their sensitivity to neonicotinoids. Another residue in loop C possibly important in determining neonicotinoids sensitivity is Ls191. Using P. pseudoannulata $\alpha1$ or $\alpha8$ co-expressed with rat $\beta2$ receptors, Meng et al. (2015) showed that chimera receptors are differentially sensitive to imidacloprid at least partly due to the difference in loop C region, equivalent to Ls184-191. Glutamate at this position, coupled with asparagine Ls148 conferred high affinity to clothianidin and imidacloprid binding in Nl$\alpha8$ rat $\beta2$ receptor in comparison to Cyrtorhinus lividipennis/rat $\beta2$, which has threonine at Ls148 and phenylalanine at Ls148 [@guo2015]. Mutation of asparagine to threonine on its own did not have marked effect on sensitivity. What is more, basic residue in loop G on the non-binding site forming interface of α-α subunits (in receptors where α: non-$\alpha$ subunit stoichiometry = 3:2) corresponding to Lys34 in LsAChBP, is a common feature among almost all insect nAChR subunits, and it is also present in human $\alpha1$, 2, 4 and 7, suggesting it may play an important role in neonicotinoid activity. Mutation of serine to arginine in chicken $\alpha7$ receptor led to enhancement of imidacloprid activity, whilst diminishing activity of acetylcholine and nicotine (@ihara2015). Finally, genetic analysis of imidacloprid-resistant strain of Nilaparvata lugens identified Y151S in loop B of Nla1 and Nla3 as a cause of this [@liu2005]. This 151 corresponds to LsAChBP H145.
<!-- Due to the absence of high resolution structures of insect nAChRs, identification of features determining high sensitivity to neonicotinoids has been hindered. @ihara2008 and @talley2008 derived crystal structures of the great pond snail (Lymnaea stagnalis) and California sea slug (Aplysia californica) AChBPs complexed with neonicotinoids (imidacloprid, clothianidin, thiacloprid), and non-selective nAChR ligands (nicotine, epibatidine and desmotroimidacloprid) to reveal differences in binding modes between the two groups of compouds. A unique electrostatic interaction between imidacloprid ring substituent (terminal oxygen of the nitro group) and basic amino acid in loop D was found. Interestingly, loop D arginine to threonine mutation naturally occurring in β1 subunit of Myzus persicae, gives rise to neonicotinoid resistance [@bass2011]. Mutation of glutamine in loop D of human $\alpha7$ to basic residue, markedly increases sensitivity of the $\alpha7$ homopentamer to nitro-containing neonicotinoids [@shimomura2002], whereas mutation of loop D threonine to acidic residues in chicken $\alpha4\beta2$ and hybrid chicken/drosophila $\alpha2\beta2$ receptor had an opposite effect [@shimomura2006]. Described mutations did not influence the efficacy of nicotinoids, suggesting this interaction is specific to neonicotinoids. Above evidence suggests that subunits containing basic residue (lysine/arginine) in loop D are important in formation of neonicotinoid-sensitive receptors. Honey bee $\alpha6-7$ and $\beta1-2$ all possess acidic residue in the exact position. However, it is likely, there are other amino acids important in nAChR-neonicotinoids interactions, some of which might be further away from the orthostatic binding pocket, allosterically influencing binding affinity. -->
## Potential neonicotinoid-sensitive insect nAChR subunits
Numerous studies were conducted aiming to identify which receptor subunits are targeted by neonicotinoids. Ligand binding studies on mammalian cell lines transfected with chimera rat $\beta2$/drosophila receptors, led to identification of three subunits conferring high neonicotinoid binding affinity: Drosophila $\alpha1$ (D$\alpha1$), D$\alpha2$ and D$\alpha3$ [@lansdell2000]. The analogues of these subunits seem to be also involved in neonicotinoid binding in other receptor subunits: $\alpha2$ and $\alpha3$ in aphid *M. persicae* [@huang1999], $\alpha1$ and $\alpha3$, $\alpha1/\alpha2$, $\alpha3$ and $\alpha8$ in brown planthopper (Nilaparvata lugens) [@liu2005; @liu2009; @yixi2009] and $\alpha1$ and $\alpha3$ in cat flea (Ctenocephalides felis) [@bass2006]. However, all of these subunits were co-expressed with rat $\beta2$, whereas in case of cat flea receptors, the ligand binding domains (LBD) was additionally fused to C-terminal transmembrane SAD motif of Drosophila $D\alpha2$. Therefore neither of these receptors represent biologically relevant protein. In addition, none of the above subunits when expressed as homooligomers in Xenopus oocyte gave rise to functional nAChR.
Potential subunits targets of neonics
Nilaparvata lugens (Nl) $\alpha1$, $\alpha2$ and $\beta1$, as well as $\alpha3$, $\alpha8$ and $\beta1$ co-assembly (the exact stoichiometry unknown) and mediate high and low affinity imidacloprid binding [@li2010], which is also present in other insects, such as *M. persicae* [@lind1998].
Locust $\alpha1$ (Schistocerca gregaria ($L\alpha1$)) expressed in Xenopus oocytes, produced low current (low Nano amp values) with nAChR-like pharmacological and electrophysiological characteristics: the currents were produced and binding of nAChRs agonists and antagonists) [@marshall1990] and sensitivity to nitromethylene neonicotinoids, [@leech1991].
To pharmacologically characterise nAChR from insect pests and other species to better understand the selective toxicity of neonicotinoids by the heterologous expression in nemotode and model organism *C. elegans*. The next section gives an overview of the *C. elegans* biology and highlights the main advantages of using this organism as a heterologous expression system.
## Overview of *C. elegans*
*C. elegans* is a transperent non-parasitic nematode, enhabiting 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 legislations. The cost of use is low, due to the cost of purchase (~$6/strain), maintanance, 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}
*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.
(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.
```{r life-cycle-label, fig.cap="(ref:life-cycle)", fig.scap='The life cycle of \\textit{C. elegans}.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/intro_2/life-cycle.jpg")
```
## Basic anatomy {#anatomy}
Anatomically an adult is about a millimeter long, rod shaped, with tapered ends. There are three major openings of the worm. Posterior there is a cuticle limned buccal cavity responsible for the initial uptake of food and its passage into the digestive tract. There is vulva opening at the ventral side of the worm, just below the midbody, from which eggs are expelled. The proctodeum (anus in hermaphrodites and cloaca in males) used for defecation is situated on the ventral side towards the posterior end of the worm. As buccal cavity, it is cuticle lined.
The worm body wall is lined by several layers. The most exterior is the cuticle. The cuticle in an exoskeleton which plays an essential role in worm’s protection from the external stimuli, as well as growth, development [@gravato-nobre2005] and locomotion [@darby2007]. Structurally, it consists of 5 distinct layers (Figure \@ref(fig:cuticle-label)). The main component is heavily cross-linked collagen, but there are also insoluble proteins such as cuticlin, modified surface glycoproteins and lipids. Glycoproteins are essential in defense mechanism and protection against the external stresses [@gravato-nobre2005; @cipollo2004; @hoflich2004; @darby2007; @partridge2008]. Directly below is an epithelium system, followed by the basal membrane resting on top of the body wall muscle.
Directly beneath is the hemolymph-filled pseudocoelom cavity. This cavity is filled with fluid - hemolymph, which transports nutrients, gases and signalling molecules. It also provides rigidity to the worm's body due to the hydrostatic pressure of the liquid within. On the basal side of the pseudocoelom cavity lay the internal reproductive and the alimentary tract.
The alimentary tract is a tubular structure running along the length of the body. Anatomically it can be divided into the buccal cavity, the pharynx, the intestine and the rectum and anus. The food particles enter, run along the alimentary tract and are excreted due to the musculature of this system. Pumping and the peristalsis of the pharyngeal and the action the enteric muscles of the and relaxation of the anal sphincter muscles. The intestine is devoid of muscle tissue.
Parallel to the alimentary tract is the reproductive system (the germ line). Hermaphrodites contain a paired, deflected into a U shape gonads, connected at the uterus. Distal end containing mitotic germ cells in a syncytium. These cells are distributed around the outer surface of the gonad tube with a common cytoplasmic core known as the rachis. As germ cells progress from the distal through the bend to the proximal end of the gonad the differentiation into oocytes occurs. The cell membrane encloses the nuclei enclosing the cytoplasmic material including protein and RNA from the rechis within [@wolke2007]. Oocytes undergoes meiotic maturation and only in the presence of sperm they mature and ovulate [@mccarter1999] into a specialised structure known as the spermatheca where fertilisation occurs. The embryo develops for the 2.5 hours in the uterus before being expelled into the external environment via the uterus.
(ref:cuticle) **Structure of the *C. elegans* cuticle.**
Transmission electron micrograph (left) and the schematic representation (right) of cross-section of the adult cuticle highlighting the major layers and constituting components. Figure taken from [@page2014].
```{r cuticle-label, fig.cap="(ref:cuticle)", fig.scap='Structure of the \\textit{C. elegans} cuticle.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/intro_2/cuticle_1.jpg")
```
<!-- The activity of the nervous system is mainly governed by neurotransmitters common to vertebrates and invertebrates: ACh, GABA, glutamate, serotonin (5-hydroxytryptamine (5-HT)) and dopamine. -->
### Behaviour as an analytical tool {#analytical_behaviour}
Over half of the century of *C. elegans* research developed a great depth of understanding of many of their simple and more sophisticated behaviors. These behaviors can be can be scored and quantified to inform on the effects of compounds or genetic altercations on worms.
An example of a well defined worm behavior is pharyngeal pumping. Pharyngeal pumping is the feeding behavior of the worm mediated by the pharynx. Successive and timed contraction-relaxation cycles of this muscular organ results in the capture, misceration and passage of the food particles down the alimentary track. The pharynx can be divided into three anatomical features: most anterior corpus, middle isthmus and posterior terminal bulb.
Pharyngeal pumping can be easily scored by counting the number of pharyngeal pumps over time to determine the effects of compounds or genetic alteration on the function of the pharynx. In addition, pharyngeal cellular assays can be performed which offer not only a greater temporal resolution of the activity of the pharynx, but also an analysis of the function of distinct anatomical features of the pharynx.
EPG (electropharyngeogram) is an extracellular electrical recording from the pharynx of the worm. It arises as a result of the flow of ions out of the worm's mouth, due to the changes in the membrane potential of the pharyngeal muscle. A single pharyngeal pump gives rise a series of electrical transients collectively called an EPG. These electrical transients are temporally defined and represents activities of distinct anatomical feature of the phayngeal muscle, namely the corpus, isthmus and the teminal bulb [@raizen1994; @franks2006].