---
nocite: | 
  @chen1997, @araujo1988, @couturier1990, @cooper1991, @lee1967, @brown1936, @mishina1986, @zirger2003, @mongeon2011, @lewis1987, @treinin1998, @richmond1999, @boulin2008, @touroutine2005, 
...

# General introduction {#generalintro}

## Selective toxicity of neonicotinoid insecticides

Selective toxicity is a key term in pharmacology and refers to the ability of a drug to explicitly act on the target. For many pest-controlling agents released into the environment, including insecticides, an ability to kill a target pest, without impacting humans, or beneficial animal species is a key determinant of their marketing success [@sanchez-bayo2018]. The understanding of selective toxicity has a long history of studies and has been an important driver in development of novel insectides [@casida1998].

### The history of insecticides

Insecticides are compounds utilised in agriculture, medicine, industry and private households to protect crops, life-stock and human health from pest infestation [@anadon2009; @dryden2009; @oberemok2015]. Until late 1800s organic, natural compounds contained within the plant or animal matter were utilised [@casida1998]. The first record of agricultural application of nicotine-containing *Tobacco* [@david1953; @steppuhn2004] dates back to 1690 [@mcindoo1943]. *Tobacco* plant was used in France, England and in the U.S. to protect orchards and trees against a wide range of pests including aphids, caterpillars and plant lice [@mcindoo1943]. *Chrysanthemum* plants containing pyrethrum were used against worms and insects in America and Europe [@elliot1995]. These treatments were however suitable only for small scale agricultural treatment, due to the limited availability.

Arsenic compounds were the earliest inorganic insecticides. Although their history dates back to 5th century [@kerkut1985], they did not gain popularity until the 19th century. Aceto-arsenite Paris Green 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 effective against pests, these substances are toxic to humans [@nelson1973; @gibb2010; @argos2010] thus their current use is marginal [@echa2017]. 

In the last century, several synthetic compounds became available, including dichlorodiphenyltrichloroethane (DDT), and members of the carbamate, organophosphate and pyrethroid class of compounds. DDT was one of the most popular insecticides in the 1900s, with the peak annual use of over 85 000 tonnes in the U.S. alone [@phsa2002]. DDT's potent insecticidal activity was discovered 60 years after its synthesis in 1874, by the Swiss chemist Paul Hermann Muller, who was later awarded a Nobel prize in Medicine “for his discovery of the high efficiency of DDT as a contact poison against several arthropods.” [@nobel2019]. DDT became commercially available in the 1940s in Europe and the U.S., and it was used to suppress potato beatles, mosquitoes, fleas and lice. Since 1970s, the use of DDT has been progressively phased out due to its propensity to bio-accumulate in the adipose tissues of animals resulting in the environmental persistence [@EUEPA1975]. 

Diminishing popularity of DDT, created a market space for organophosphates, carbamates and pyrethroids (Table \@ref(tab:insecticidegroups)). By the 1990s, the respective market share of members of these three classes of insecticides was: 43%, 15% and 16% and the annual sales of 3.42, 1.19, bn and 1.17 billion Euro, respectively [@jeschke2011]. The main issue associated with the use of organophosphates and carbamates is their ability to cause serious human poisoning, which can lead to death [@king2015]. The lack of selectivity combined with increasing resistance [@bass2014] 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.

```{r insecticidegroups, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
 library(dplyr)
 insecticide_groups <- data.frame(
   Class = c("Organophosphates", "Carbamates", "Pyrethroids"),
   Chemicals = c("parathion, malathion, azinphosmethyl", "aldicarb, carbamyl, carbofuran", "allethrin, cypermethrin"),
   Mode = c("Acetylcholinesterase\ninhibitor", "Acetylcholinesterase\ninhibitor", "Voltage gated\nsodium channel blocker"))
   
 insecticide_groups %>%
    mutate_all(linebreak) %>%
 kable("latex", align = "l", booktabs = TRUE, escape = F,
       col.names = linebreak(c("Class", "Chemical", "Mode of\naction")),
       caption = 'Synthetic insecticides') %>%
   kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position")
```

\newpage

### Structural diversity 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 are unstable in light. The latter however is successfully used in veterinary medicine as an external parasite treatment for cats and dogs.

To solve the issue of photo-instability, nitromethylene group (CCHNO~2~) was replaced by nitroguanidine (CNNO~2~) and cyanoamidine (CNCN) (Figure \@ref(fig:neonics-structure-label); @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, 6 most commonly used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include confidor, admire and advantage. Together with thiacloprid (calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (actara) is produced by Syngenta, Clothianidin (poncho, dantosu, dantop) and Nitenpyram (capstar) by Sumitomo Chemical, acetamiprid (mospilan) by Certis, whereas dinotefuran (starkle) by Mitsui Chemicals company. Last neonicotinoid, dinotefuran, was launched in the EU in 2008.

Research into novel neonicotinoids continues [@shao2013]. In the last decade, several novel insecticides have been characterised and approved for use in the EU. Sulfoxafrol [@zhu2011; @eu2019a] and flupyradifurone [@nauen2015; @eu2019b] have been classified as representatives of new chemical classes, namely sulfoximines and butenolides. However, due to their mode of action and similar biochemical properties, some argue that they are in fact neonicotinoids, whereas their mis-classification has been deliberate to avoid association with neonicotinoids [@pan2019].

(ref:neonics-structure) **Development and chemical structures of the synthetic insecticides, the 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 structurally related to nicotine (shown in top right corner) and 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 = '90%', echo = FALSE}
knitr::include_graphics("fig/general_intro/png/neonics_structure.png")
```

### Economical status of neonicotinoids ###{#economicalstatus}

The use of neonicotinoids in agriculture has been increasing steadily since their launch in the early 1990s. By 2008, they became major chemicals in the agriculture, replacing organophosphates and carbamates [@jeschke2011]. Continual increase in popularity of neonicotinoids is reflected in the total usage data. In Great Britain, the yearly use of neonicotinoids increased by over 10-fold from 10 tonnes/year in 1996 to over 105 tonnes/year in 2016 [@fera2019]. Similar trends are observed in the U.S. [@usgs2019], Sweden and Japan [@simon-delso2015]. Continual increase in usage coincides with the rise in their economical impact. In 2008, the estimated global market value of neonicotinoids was 1.5 billion dollars [@jeschke2011]. This increased to 3.1 billion dollars in 2012 [@bass2015]. 

The widespread usage and monetary value of neonicotinoids is a reflection of their many advantages. 

\newpage

### Physicochemical properties of neonicotinoids grant versatile methods of application  ###{#physchem}

One of the major benefits of neonicotinoids are their physical and chemical profiles (Table \@ref(tab:properties)). Due to relatively high water solubility, neonicotinoids act as systemic insecticides [@westwood1998]. This means that once applied on crops, they dissolve in the available water and are taken up by the developing roots or leaves. Upon plant entry, they are then distributed to all parts of the plant [@westwood1998; @stamm2016], providing protection against herbivorous pests [@stamm2016]. This 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. Neonicotinoids 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. They are not volatile, therefore can be also applied as spray. This method is used in the gardens 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 of animals [@turaga2016]. However, moderate water solubility combined with low lipophilicity means they may have a potential to accumulate in water.

```{r properties, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
 library(dplyr)
 properties <- data.frame(
   Compound = c("Nitenpyram", "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 = 'Physicochemical properties of neonicotinoids',
       ) %>%
   kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
   add_footnote(notation = "none", "log Pow = octanol/water partitioning, 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)
```

\newpage

### Neonicotinoids are highly potent against insect pests ###{#potentpests}

<!-- look at this paper to see the symptoms of imi exposure on insects  -->
<!-- @sone1994 -->
Neonicotinoids are highly potent against insect pests, (Table \@ref(tab:toxallanimal)), including a wide range of piercing-sucking pests such as cotton and peach aphids (*Aphis gossypii, A. gossypii* and *Myzus persicae, M. persicae*) [@nauen1996; @mota-sanchez2006; @bass2011], house- (*Malus domestica, M.domestica*) and may-flies (*Epeorus longimanus, E. longimanus*) [@tomizawa2000; @alexander2007] as well as planthoppers (*Nilaparvata lugens, N. lugens*) [@zewen2003]. Their LC~50~ (the concentration needed to kill 50% of pests) in generally in the region of 2 $\mu$M. Although all neonicotinoids are highly effective against insect pests, their potency depends on the chemical structure. The rank order of insecticidal potency on the cotton aphid *A. gossypii* and the Colorado potato beetle, *Leptinotarsa decemlineata* (*L. decemlineata*) was clothianidin > nitenpyram = thiacloprid, suggesting nitroguanidines are generally more potent than nitromethylenes and cyanoamidines [@shi2011; @mota-sanchez2006]. 

The potency also depends on the route of exposure. LC~50~s are lower upon systemic or oral administration in comparison to the topical exposure [@alexander2007]. Imidacloprid injected into the abdomen of American cockroaches *Periplaneta americana* (*P. americana*), killed 50% of animals at 1 nM [@ihara2006]. Concentrations of 285.49 nM and 1.83 $\mu$M were required to observe the same effect upon oral or contact exposure, respectively in the peach aphid *M. persicae* [@nauen1996]. Effective doses obtained from oral and topical studies are most relevant, since these are the two main routes of exposure of pests in the agriculture. 
The LC(D)~50~ values of neonicotinoids are at least 6-fold higher than those of structurally related nicotine, highlighting the superiority of neonicotinoids as pest controlling agents. 

\newpage
```{r toxallanimal, echo=FALSE, warning = FALSE, message = FALSE}
library(kableExtra)
library(dplyr)
footnotea <- "References (Ref) 16: Shi et al. 2011, 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"

toxic <-data.frame (
  Drug = c("Thia", "Clo", "Nit", "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", "Thtx", "Clo", "Clo", "Clo", "Clo", "Clo", "Clo", "Imi", "Imi", "Clo", "Clo", "Clo", "Imi", "Ace", "Nit", "Clo", "Thia", "Thia"),
  Species = c("\\textit{A. gossypii}", "\\textit{A. gossypii}", "\\textit{A. gossypii}",  "\\textit{M.persicae}", "\\textit{M.persicae}", "\\textit{M.persicae}", "\\textit{M.persicae}", "\\textit{M.persicae}", "\\textit{M.persicae}","\\textit{M.persicae}", "\\textit{M.persicae}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{L. decemlineata}", "\\textit{N.lugens}","\\textit{M. domestica}", "\\textit{M. domestica}",  "\\textit{E. longimanus}", "\\textit{E. longimanus}", "\\textit{A. mellifera}", "\\textit{A. mellifera}", "\\textit{A. mellifera}","\\textit{A. mellifera}","\\textit{A. mellifera}", "\\textit{C. virginianus}", "\\textit{C. virginianus}", "\\textit{A. platyrhynchos}", "\\textit{M. musculus}", "\\textit{O. mykiss}", "\\textit{L. macrochirus}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", " \\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{E. fetida}", "\\textit{M. incognita}"),
  Common = c("Cotton aphid", "Cotton aphid", "Cotton aphid",  "Green peach\naphid", "Green peach\naphid", "Green peach\naphid", "Green peach\naphid", "Green peach\naphid", "Green peach\naphid","Green peach\naphid", "Green peach\naphid", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Colorado\nbeetle", "Brown\nplanthopper","Housefly", "Housefly",  "Mayfly", "Mayfly", "Honey bee", "Honey bee", "Honey bee","Honey bee","Honey bee", "Bobwhite quail", "Bobwhite quail", "Mallard duck", "Mouse", "Rainbow trout", "Bluegill", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Redworm", "Root-knot\nnematode"),
  Taxon = c("Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest",  "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npest", "Insect\npollinator", "Insect\npollinator", "Insect\npollinator", "Insect\npollinator", "Insect\npollinator", "Bird", "Bird", "Bird", "Mammal\n", "Fish\n", "Fish\n", "Earth worm", "Earth worm","Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Nematode"),
  LD50 = c("-", "-", "-", "-", "-", "-", "-",  "-", "-", "-", "-", "0.35 ng/mg", "0.05 ng/mg", "6.8 ng/beetle", "0.34 ng/beetle", "0.20 ng/beetle", "0.18 ng/mg", "0.15 ng/mg", "0.14 ng/mg", "0.82 ng/mg", "3 ng/mg", ">50 ng/mg", "-", "-", "-", "0.81 ng/mg", "0.81 ng/mg", "0.44 ng/mg", "0.24 ng/mg", ">200 mg/kg\n(acute)", ">5040 mg/kg\n(5 days)", ">5230 mg/kg\n(5 days)", "389-465 mg/kg", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-"),
  LC50 = c("9.35 $\\mu$M", "7.29 $\\mu$M", "9.12 $\\mu$M", "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\n(24 hrs)", "2.54 nM\n(96 hrs)", "6.88 $\\mu$M", "-", "-","-","-","-", "-", "-", "-", "424.51 $\\mu$M", "468.60 $\\mu$M", "4.81 $\\mu$M\n(24 hours)", "2.74 $\\mu$M\n(48 hours)", "62.08 $\\mu$M\n(14 days)", "24.24 $\\mu$M\n(7 days)", "24.27 $\\mu$M\n(14 days)", "11.93 $\\mu$M\n(14 days)", "12.08 $\\mu$M\n(14 days)", "26.75 $\\mu$M\n(14 days)", "3.72 $\\mu$M\n(14 days)", "10.60 $\\mu$M\n(14 days)", "143. 24 $\\mu$M\n(6 hours)"),
  Bioassay = c("Topical", "Topical", "Topical", "Topical", "Oral", "Topical", "Oral", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "?", "?", "Topical", "Topical", "Oral", "Oral", "Topical", "Topical", "Topical", "Oral","Oral", "Oral", "Oral", "?", "?", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical", "Topical"),
  Ref = c("16", "16", "16",  "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 %>%
      mutate_all(linebreak) %>%
      kable("latex", align = "l", escape = F, booktabs = TRUE, longtable = TRUE,
            col.names = linebreak(c("Drug", "Species", "Common\nname", "Taxon", "LD50", "LC50", "Bioassay", "Ref")),
      caption = 'Toxicity of nicotine and neonicotinoids') %>%
  kable_styling(font_size=9, position = "center", full_width = FALSE, latex_options = c( "hold_position", "repeat_header")) %>%
  footnote(general = footnotea,
                threeparttable = TRUE)
```

\newpage

### Neonicotinoids are selectively lethal to insect pests ###{#seltox}

The key determinant of success of agrochemical compounds is their ability to selectively target pests over non-target species. Neonicotinoids are generally effective at ~ 2 $\mu$M concentrations against piercing-sucking pest infestations, whereas their LD~50s~ are in the region of 0.2 - 0.3 ng/mg of body weight [@mota-sanchez2006; @zewen2003; @tomizawa2000; @alexander2007]. The LC(D)~50~ values for non-target species are at least 2 times higher (Table \@ref(tab:toxallanimal)). Honeybees (*Apis mellifera, A.mellifera*) are among the most susceptible non-targets, with the average LC~50~ and LD~50~ values for imidacloprid of 7.04 $\mu$M and 4.5 ng per mg of body weight, respectively [@cresswell2011]. Some studies report high potency of neonicotinoids on earth worms, with the LC~50~ as low as 2.74 $\mu$M on redworm *Eisenia fetida* (*E. fetida*) [@luo1999]. Fish and birds are hundred fold less susceptible [@decant2010], whereas mammals are the least susceptible with LD~50~ doses higher than 130 mg/kg of body weight [@decant2010; @legocki2008]. This differential susceptibility between target and non-target species, is expected to enable an environmental release of neonicotinoids at concentrations which will exterminate pests without killing the non-targets.

Indeed, field realistic concentrations of neonicotinoids are higher than those causing lethality of the most susceptible species of honey bee and worms. Residues of neonicotinoids can be found in the nectar, wax and pollen and nectar, with the highest concentration generally present in the former [@goulson2013]. @cresswell2011 determined that imidacloprid is present in most commonly bee-consumed nectar at 2.3 - 20 $\mu$M. He also estimated that the average realistic amount of imidacloprid in a nectar load is 0.024-0.3 ng. This is higher than the reported honeybee LC~50~ and LD~50~ values of 7.04 $\mu$M and 4.5 ng, respectively [@cresswell2011]. 

The concentration of neonicotinoids in soils with several years of history of treatment by seed coating were also investigated. Samples were collected 10 months after sowing [@botias2015] just before [@jones2014; @schaafsma2016] or after planting [@perre2015]. The average reported concentrations of neonicotinoids in the centre of the field are in the region of 20 $\mu$M, which is higher than the concentrations effective against earth worms and nematodes; the LC~50~ against the most susceptible species is 2.74 $\mu$M [@luo1999]. A differential between the concentration of neonicotinoids in the field and the LD~50~ values, suggest that neonicotinoids are not expected to kill bees or worms, however, a substantial body of evidence from lab- and field- based experiments suggest that they can impair on the behaviour and the cognitive function of these animals. 

<!-- [@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 degradation half-life (TD50) of clothianidin was estimated at 0.4 years 9 (146 days). It was also concluded that 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. -->

### Sub-lethal effects of neonicotinoids on non-target species ###{#sublethal}

#### Effects on insect pollinators ###{#sublethalbees}

Pollinating services are provided by many species of bees, flies, beetles and bats [@thapa2006]. Eighty percent of the total pollinating activity is carried out by bees [@thapa2006]. There are over 20 000 species of bees, 267 species live in the UK [@breeze2012a]. Among them are honey bees (*A. mellifera*), bumblebees and over 220 species of solitary bees. Honeybees and bumblebees served as platform to determine toxic effect of neonicotinoids on biological pollinators. 

##### Reduced olfactory learning and memory

Honeybees are social insects, living in colonies where a clear division of labor exists. Worker bees account for up to 95% of the entire colony [@sagili2011]. These non-reproductive females are responsible for finding, collecting and transporting nectar or pollen from the flowering plants to the hive. Their ability to process, learn, memorise sensory cues and navigate through the environment is crucial for the survival and overall success of the entire hive. It is these essential processes that are disrupted by neonicotinoids. Bees exposed to 93 nM of imidacloprid in the sugar solution showed reduced ability to olfactory learn, as showed by the Proboscis Extension Reflex (PER) [@decourtye2004] - a paradigm used to measure olfactory learning of bees [@takeda1961]. Imidacloprid also compromised foraging activity of honeybees [@decourtye2004; @gill2012] and bumblebees. Four-week exposure of early-developmental stages to imidacloprid at 23 nM in pollen reduced the foraging efficiency and duration [@gill2012]. Neonicotinoids at low nM concentrations, also reduced the number of bees returning to hives [@henry2012; @feltham2014]. 

<!-- #### Learning and memory  -->

<!-- Honeybees exposed to a , reduced  Reducing homing rates were also noted in a study conducted by @schneider2012, where bees were exposed to clothianidin, thiamethoxam and imidacloprid at doses ranging from 0.05 ng/bee to 6 ng/bee.  -->
<!-- <!-- Reduced response in conditioning assay was noted in honey bees treated with imidacloprid at 10 and 100 nM chronically (4 days), which were then allowed to recover for three days [@williamson2013].  --> 

##### Impaired reproduction

Neonicotinoids have been also shown to negatively impact on various aspects of bees' fecundity. Reproduction of bees is performed by a single member of the colony - the queen. She lays fertilised and unfertilised eggs into cells of the comb. These eggs develop into larva, pupa and adult male drones and female workers. Fourteen day exposure of bumblebees to imidacloprid at 2, 4 and 23 nM, increased the number of empty pupal cells [@whitehorn2012]. Imidacloprid has been shown to reduce the total size of treated colonies, reduce the brood production [@laycock2012] and the number of born queens [@whitehorn2012] and workers [@gill2012] of bumblebees. Exposure of drones to thiamethoxam at 15.5 nM and clothianidin at 6 nM led to shortening of life-spam and hindered sperm vitality and quantity [@straub2016]. Although these effects were observed in the lab- and semi- lab conditions, the negative impact of neonicotinoids was also seen in the 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 bees and honeybees, restricted their use in Europe in 2013 [@eucomission2013] and is likely to lead to a complete ban of neonicotinoids in the future [@efsa2018]. 

#### Effects of neonicotinoids on worms ####{#effectsofneonicsonworms}

Worms can be divided into several phyla, including Platyhelminthes (flatworms), Annelida (segmented worms) and Nematoda (roundworms, pinworms, etc.). These animals can live on land, in the ocean, in freshwater, in or on animals and plants. They can be further divided into parasitic and free-living, non-parasitic worms. In the soil, non-parasitic earth worms and nematodes have an important biological role whereby they are a significant biomass contributors and nutrient cyclers [@ingham1985; @neher2001; @lecomte-pradines2014] (Section \@ref(ecologoicalroleofwormschapter3)). They have also an important role in shaping the soil structure [@blouin2013]. These functions are heavily reliant on worms ability to burrow or move in the soil, feed and reproduce [@medina-sauza2019], however it is these behaviours that have been shown to be impacted by neonicotinoids. 

##### Earth worms

Clothianidin and thiacloprid at concentrations $\ge$ than 1.2 $\mu$M and the EC~50~ of 5.1 $\mu$M and 3.4 $\mu$M, respectively reduced the reproductive potential of redworm *E. fetida*, as measured by the cocoon production [@gomez-eyles2009]. Neonicotinoids showed a negative impact on the reproduction of other species, including *Lumbricus rubellus* (*L. rubellus*) [@baylay2012], *Dendrobaena octaedra* (*D. octaedra*) [@kreutzweiser2008] and *Eisenia andrei* (*E. andrei*) [@alves2013]. Reduction of body weight of *E. fetida* and *D. octaedra* were observed after a 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], whereas 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]. 

##### Soil nematodes #####{#soilnematodesneonicstoxicity}

Neonicotinoids also induce sublethal effects on the the free-living nematode *C. elegans*. Thiacloprid and imidacloprid impaired on the reproduction of *C. elegans* with EC~50~ of 1.14 nM and 2.09 mM, respectively [@gomez-eyles2009]. Thiacloprid at 37 nM has an effect on chemosensation, whereas at 18 $\mu$M it impairs motility of this free living nematode [@hopewell2017]. Impaired motility of *C. elegans* in response to $\ge$ 120 $\mu$M imidacloprid was also recorded [@mugova2018]. Taken together, neonicotinoids have sublethal effects on earth worms and soil nematodes at concentrations as low as nM. 
Most of the doses effective against worms are higher than the average doses of neonicotinoids in the field. However, the presence of clothianidin, imidacloprid and thiamethoxam has been detected at lower than average levels, such as 80.10 nM for imidacloprid, 23.01 nM for imidacloprid and 68.56 nM for thiamethoxam [@jones2014]. This suggests that the environmentally relevant concentrations of neonicotinoids may negatively impact on the the well-being of soil dwellers. 

<!-- Data regarding toxicity of neonicotinoids on soil worms and nematodes focuses on earthworm *Eisenia fetida* (*E. fetida*, redworm). The toxicity is assessed either following treatment in solution or in the artificial soils. In solution, the LC50 of imidacloprid after 24 hour exposure is 62.08 $\mu$M [@luo1999]. This increases to 24.24 $\mu$M after 48 hour exposure [@luo1999]. Clothianidin is less potent; its LC50 after 14 days is 24.27 $\mu$M [@decant2010]. -->

#### Effects of neonicotinoid on birds

Environmentally relevant concentrations of neonicotinoids may also have a negative effect on birds [@hallmann2014]. In particular, granivorous and insectivorous birds may be at risk, should they consume neonicotinoid-contaminated seeds and/or insects [@goulson2013]. Environmental neonicotinoids may impair their  migratory ability [@eng2017] and negatively impact on their growth and reproduction [@sanchez-bayo2016].

The environmental ecotoxicity of neonicotinoids highlights the importance of selective toxicity of agrochemical compounds in successful pest management programmes. The development of new insecticides, effective against pest and not beneficial insects or other species requires a detailed knowledge of their mode of action.

\newpage

## Neonicotinoids act by binding to nicotinic acetylcholine receptors (nAChR)

<!-- ### Nicotinic acetylcholine 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") -->
<!-- ``` -->

### nAChR structure ###{#structure}

Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channels which are found in a diversity of species from bacteria to human. They are the representatives of the Cys-loop superfamily of channels which also include $\gamma$ -aminobutyric acid (GABA) type A receptors, 5-hydroxytryptamine type-3 receptors (5-HT3), and glycine receptors. Structural studies of the nAChRs from the muscle of the electric fish *Torpedo* (Figure \@ref(fig:structure-nachr-label)a) shed light on the the stoichiometry, the shape and the size of Cys-loop receptors.

The identity of the neuromuscular junction (NMJ) nAChR was first investigated using indirect, biochemical approaches. Membrane bound NMJ receptors were isolated by *in-situ* cross-linking with a radiolabelled antagonist and a subsequent purification. sodium dodecyl sulfate (SDS) -resolved fragments pattern suggesting the pentameric nature of these receptors [@hucho1986; @schiebler1980] of the total size 270 000 kDa composed of 4 different subunits namely $\alpha$, $\beta$, $\delta$ and $\gamma$ arranged into a pentamer. The SDS- polyacrylamide gel electrophoresis (PAGE) pattern and the analysis of nAChR complexes purified with the use of non-denaturing buffer led to a suggestion that the stoichiometry is: $\alpha1$, $\beta1$, $\delta$, $\alpha1$, $\gamma$ (clockwise) [@reynolds1978]. Heterologous expression in *Xenopus* oocytes confirmed that 4 subunits are needed to achieve expression. In the absence of any other one of the subunits, the responses to acetylcholine (ACh) were either absent or greatly reduced, therefore 4 subunits are required for the normal function of this protein [@mishina1984]. 

The stoichiometry and structural details of muscle type nAChRs were confirmed by more direct structural approaches: cryo- and electron- microscopy. 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. Constituent 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 pore-forming M2 helices [@imoto1986; @hucho1986] from the hydrophobic environment of the bilayer. 

(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-terminal $\alpha$-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 neighboring subunits (A-B, B-C, C-D, D-E and E-A), named the principle and the adjacent components, respectively. Top view of the molluscan AChBP (PDB:1I9B) with amino acids forming the agonist binding site in ball and stick representation (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")
```

\newpage

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 $\alpha$E241 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. 

### Model of the nAChR binding site ###{#modelodnachbinding}

Determination of the crystal structure of the molluscan acetylcholine binding protein [@brejc2001, Figure \@ref(fig:binding-pocket-label)b and c)] 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 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 the great pond snail *Lymnaea stagnalis* (*stagnalis* are discussed.

(ref:binding-pocket) **The ligand binding domain of acetylcholine binding protein.** Agonist binds to the loops situated in the adjacent subunits of the nAChR. In muscle type receptor, there are 2 binding sites, and there are 5 in homopentameric receptor (a). The ligand binding pocket of the AChBP (PDB:1I9B) is formed from loops of the neighboring 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")
```

### Agonist binding site of nAChRs ###{#bindingsite}

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 receptor, it constitutes of $\alpha$ and non-$\alpha$ subunit contributions, whereas in homopentameric or $\alpha$ heteropentameric receptors it is made up of neighboring subunits. The principal, $\alpha$-subunit site subsides 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 originating 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. 

### Pharmacophore of nAChR agonists ####{#pharmacophore}

Crystal structure of the AChBP bound to acetylcholine, carbamylcholine, nicotine [@celie2004] and its analogue epibatidine [@hansen2005] provided some general features of the nAChR binding pocket. More recently, structures of mammalian receptors: $\alpha9$ [@zouridakis2014] bound to methyllycaconitine, the artificially expressed $\alpha2$ extracellular domain bound to epibatidine [@kouvatsos2016] and $\alpha4\beta2$ receptor bound to nicotine [@morales-perez2016] have been obtained. These structures provide details of how structurally varied agonists bind to nAChRs. 

The agonist binding site is buried on the interface of the neighboring subunits. They are stabilised in the binding pocket by 5 conserved aromatic residues from A, B and C loops of the principal site (known as the aromatic box), which engulf the cationic atom of the quaternary ammonium atom of bound agonist. There are two major and conserved features: cation-$\pi$ interaction and hydrogen bond. 

Cation -$\pi$ interactions are formed between the cationic nitrogen and aromatic 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 structures, 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 lower efficacy and affinity. Heterologously expressed $\alpha7$ are activated with choline with the EC~50~ between 0.4 and 1.6 mM, whereas the EC~50~ of nicotine is between 49 and 113 $\mu$M [@wonnacott2007]. Radiolabelled studies report up to 500 times lower binding affinity of choline in comparison to nicotine [@wonnacott2007].

Cation-$\pi$ interactions and a hydrogen bond are the staple features of the ligand-receptor interactions, however there are also some less conserved characteristics. For example, in AChBP-nicotine structures, there is a hydrogen bond between cationic nitrogen of the agonist and the carbonyl of TrpB in the principal site of the receptor [@celie2004]. Similarly, in human $\alpha2$ structures a hydrogen bond between the cationic nitrogen of apibatidine and carbonyl of TrpB or Tyr in loop A is formed [@kouvatsos2016]. In contrast, cationic 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) (a). Interactions with the receptor based on the crystal structure of the AChBP and nicotine (PB:1UW6) (b). Image b) is taken from @blum2010.

```{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")
```

### Pharmacophore of neonicotinoids ###{#pharmacophoreofneonics}

Structure of AChBP proved to be also valuable in determining structural elements which may account for neonicotinoids’ selectivity. @ihara2008; @talley2008; @ihara2014 derived crystal structures of the great pond snail (*L. stagnalis*, Ls) and California sea slug (*Aplysia californica, A. californica*) AChBP complexed with 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 (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:imi-binding-label)).

Structures of wild-type and mutant AChBP with increased affinity to neonicotinoids revealed no differences in the interactions between imidacloprid, clothianidin and thiacloprid (Figure \@ref(fig:all-neonics-binding-label)) [@ihara2008; @talley2008; @matsuda2009; @ihara2015]. Thus, to describe the differences between neonicotinoids and nicotinoids, crystal structures of *L. stagnalis* AChBP complexed with nicotine and imidacloprid are compared (Figure \@ref(fig:imi-binding-label)). The positioning of the pyridine ring of imidacloprid and nicotine is virtually the same. The nitrogen forms identical interactions: hydrogen bond with the amide group of Met114 and carbonyl group of Leu102 of loop E, via water molecule [@celie2004; @ihara2008; @talley2008]. In addition, chlorine atom of imidacloprid makes van der Waals interactions with oxygen of Ile106 and oxygen of Met116 of AChBP [@talley2008].

Regarding 5-membered ring interactions, in nicotine-bound structures, the cationic nitrogen forms 3 interactions when bound to AChBP: the cation-$\pi$ with the ring of Trp143 (TrpB), the hydrogen bond with the backbone carbonyl of TrpB [@celie2004], as well as the cation-$\pi$ interaction with Tyr192 in loop A [@matsuda2009]. In imidacloprid bound structures, the ring stacks with aromatic residue Tyr185 of loop C [@ihara2008]. These stacking interactions result in the formation of CH-$\pi$ interactions between the methyline bridge (CH~2~-CH~2~) of imidacloprid and TrpB. All residues described so far are conserved in other agonist-bound nAChR structures, 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 (NO~2~) of imidacloprid bridges to glutamine of loop D (Gln55) via a hydrogen bond. This interaction was also seen in thiacloprid bound AChBP and in the Gln55Arg mutant of AChBP bound to clothianidin [@ihara2014]. It is interesting that in some nAChR subunits, such as *M. pyrsicae* $\beta1$, honeybee  $\beta1-2$ and $\alpha7$, 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/$\pi$ hydrogen bond interactions between the ring and the protein. In contrast, other subunits such as human $\alpha7$ or *C. elegans* ACR-16 and EAT-2 contain either acidic or polar amino acids in the exact position, repulsing or forming no electrostatic interactions with imidacloprid, which could at least in part explain low sensitivity of nematodes and mammals to neonicotinoids (Section \@ref(soilnematodesneonicstoxicity)).

(ref:imi-binding) **Pharmacophore of nicotine and imidacloprid**. Schematic representation of the agonist binding site of acetylcholine binding protein, highlighting residues interacting with nicotine and imidacloprid. 

```{r imi-binding-label, fig.cap="(ref:imi-binding)", fig.scap= "Pharmacophore of nicotine and imidacloprid", fig.align='center', out.height="70%", echo=FALSE}
knitr::include_graphics("fig/general_intro/png/nicotine_imidacloprid_structure.png")
```

Analysis of the structure of Gln55Arg AChBP mutant complexed with neonicotinoids revealed another residues with a potential to confer high binding affinity of these compounds. Basic residue of loop G, namely Lys34, forms electrostatic interaction with the NO~2~ group of clothianidin and CN group of thiacloprid, but does not interact with imidacloprid (Figure \@ref(fig:all-neonics-binding-label)) [@ihara2014]. 

(ref:all-neonics-binding) **Pharmacophore of neonicotinoids**. Schematic representation of the agonist binding site of acetylcholine binding protein, highlighting residues interacting with imidacloprid, thiacloprid, thiacloprid and nitenpyram. For nitenpyram, the interactions are predicted based on other structures.

```{r all-neonics-binding-label, fig.cap="(ref:all-neonics-binding)", fig.scap="Pharmacophore of neonicotinoids", fig.align='center', echo = FALSE, }
knitr::include_graphics("fig/general_intro/png/binding_all_neonics.png")
```

#### Selectivity of neonicotinoids

Based on the structural data, it has been proposed that the basic residue in loop D and G interacting with the nitro or cyano group of neonicotinoids is important in confirming neonicotinoid selectivity in insect nAChR subunits. This is supported by genetic studies. Loop D arginine to threonine mutation naturally occurring in $\beta1$ subunit of peach aphid *M. persicae*, and cotton aphid *A. gossypii* [@hirata2015; @hirata2017; @bass2011] gives rise to neonicotinoid resistance. Additionally, @shimomura2002 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, 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. In addition, double mutant of avian $\alpha7$ nAChR in which amino acids equivalent to Gln55 and Lys34 were mutated to basic residues showed increased binding affinity of thiacloprid and clothianidin, but not nicotine or acetylcholine [@ihara2014], providing further evidence that these residues are important in conferring high binding affinity of neonicotinoids. 

Genetic studies identified other amino acids of potential importance. Imidacloprid-resistant strain of planthopper *N. lugens* has been found to have Y151S mutation in loop B of $\alpha1$ and $\alpha3$ nAChR subunits [@liu2005]. This residue corresponds to LsAChBP H145 of the loop B. Loop B, D and G originate from the complementary site, but the principal site may also play a role. Studies on *Drosophila*/chicken $\alpha2\beta2$ hybrid and chicken $\alpha2\beta4$ receptors showed that the presence of nonpolar proline in YXCC motif of loop C enhances affinity, whereas mutation of proline to glutamate markedly reduces affinity of neonicotinoids to these receptors [@shimomura2005]. The importance of C-loop regions was also demonstrated by @meng2015 who showed that chimera receptors are deferentially sensitive to imidacloprid at least partly due to the difference in loop C region, equivalent to Ls184-191. 

Taken together, AChBP is a useful model in which potential molecular determinants of neonicotinoid-selectivity were investigated. Of particular interest is basic residue in the principal site of the biding pocket, however many other amino acids, including those in the complementary binding site and those away from the binding site may also play a role. This highlight the complex nature of the receptor-agonist binding and creates a challenging environment for the *in-silico* structure-based design of novel neonicotinoids.

## Neonicotinoids act on the cholinergic neurotransmission as a nicotine mimic 
### Cholinergic system in insects

Cholinergic neurotransmission is the process of signal propagation between neurons at the synapse. Cholinergic synapse is characterised by the presence of several proteins which mediate the breakdown, the synthesis, the processing and the function of the neurotransmitter ACh (Figure \@ref(fig:cholineric-synapse-label)).

Choline acetyltransferase (ChAT) is an enzyme synthesising ACh [@greenspan1980b], by a transfer of acetyl-CoA onto choline. There are at least two isoforms in *Drosophila*, which are produced by alternative splicing from the ChAT gene [@slemmon1982]. One is membrane bound, whereas the other is soluble [@pahud1998]. A soluble isoform of ChAT was also isolated from the locust *Schistocerca gregaria* (*S. gregaria*) [@lutz1988]. In *Drosophila*, the soluble isoform performs the majority of enzymatic activity [@pahud1998]. 

Vesicular acetylcholine transferase (VAChT) mediates ATP-dependent transport [@varoqui1996], which packs ACh into the synaptic vesicles for release [@song1997]. In *Drosophila*, a single VAChT gene was identified [@kitamoto1998].

Acetylcholinesterase (ACE) is a soluble enzyme that catalyses breakdown of ACh [@chao1980; @hsiao2004]. In the fruit fly *Drosophila melanogaster* (*Drosophila*), it is encoded by the Ace locus [@hall1976]. Acetylcholinesterase is a homodimer covalently bonded by the disulphide bridge [@chao1980; @hsiao2004]. Each monomeric subunits is ~67 kDa, folded into 4-helix bundle [@harel2000]. 

Insect nAChRs are expressed at the post-synaptic membrane [@kreissl1989; @gu2006] and mediate fast synaptic transmission [@callec1973]. Upon binding of neurotransmitter acetylcholine, nAChRs open, allowing the flux of cations down their concentration gradient [@goldberg1999], leading to depolarisation of the post-synaptic neuron and signal propagation. 


<!-- The function and properties of these receptors were studied using mammalian and amphibian muscle preparations. -->

<!-- In 1930s, @brown1936; @bacq1937 demonstrated that the application of acetylcholine, as well as nicotine and choline to the isolated mammalian muscle leads to sustained contraction, as showed by the increase in the muscle tension. The muscle contraction was associated with an increase in the frequency of the action potential firings [@brown1936] and the depolarisation of the end-plate [@katz1957]. Acetylcholine-evoked responses could be inhibited by pre-incubation with several compounds, including snake venom proteins, $\alpha$-bungarotoxin [@chang1963].  -->

<!-- Prolonged exposure to high concentration of agonist has a secondary effects on the muscle: desensitisation [@katz1957]. Desensitisation is a post-contraction period, after the removal of the agonist, at which the muscle is relaxed and a subsequent contraction cannot be elicited [@thesleft1955]. Recovery from desensitisation typically lasts few seconds after the agonist removal [@bouzat2008], however the duration varies depending on the receptor, the compound [@briggs1998] and its concentration [@gerzanich1994]. Full recovery may not occur or may be slower if the receptor are exposed to the large doses of agonist for a prolonged time [@katz1957]. -->

<!-- Biochemical, electrophysiological, genetic and pharmacological approaches were utilised to identify the role of nAChRs in neurotransmission. nAChRs were solubilised and purified from the electric organ of the *Torpedo* and reconstituted into liposomes [@anholt1982]. In the presence of acetylcholine and other agonists, the ionic current was elicited, confirming nAChR is an ion channel [@anholt1982]. Acetylcholine evoked activation ans desensitisation of nAChRs, which correspond to the effects in the muscle, provided evidence that nAChRs mediate fast synaptic transmission at the cholinergic synapse.  -->

<!-- Kinetic properties of nAChRs were studies with the patch clamp technique. Patch clamp is a technique which enables for the resolution of agonist-evoked responses at a single receptor level [@colquhoun1981; @colquhoun1985]. In response to acetylcholine the channel switches between active and inactive form, with the active form interrupted by the short-lived channel closing bursts. Temporal characterisation revealed the average duration of each event. The receptor remains opened for 1.4 ms; this is interrupted by channel closing bursts of 20 $\mu$s which occur at a frequency of 1.9 closures/opening burst. The mean period between the successive channel openings is 342 ms. Further experiments provided the showed that channel opening is not an all or nothing event. Instead, a channel exhibits multiple conductance states, one on which it is fully opened, named a full conductance state (i.e. the active form), and one in which the channel is partially opened, named the sub-conductance state [@colquhoun1985]. The fine structure of full conductance states (or opening bursts) and sub-conductance states varies depending on the agonist used and a receptor protein [@colquhoun1985; @nagata1996; @nagata1998].  -->

(ref:cholineric-synapse) **Enzymes transporters and receptors at the cholinergic synapse.** Upon release into the synaptic cleft, acetylcholine binds to nicotinic acetylcholine receptors (nAChRs) for signal propagation. To prevent overstimulation of the post-synaptic neuron, acetylcholine is broken down to choline and acetate by acetylcholinesterase (AChE). Choline is then taken up to the pre-synapse by a choline transporter (ChT). The acetyl group in transferred onto choline to produce acetylcholine. This reaction is catalysed by choline acetyltransferase (ChAT). Generated acetylcholine is pumped into the synaptic vesicle by the vesicular acetylcholine transporter (VAChT).

```{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_with_enzymes.png")
```


<!-- Development of molecular cloning technique enabled for expression and kinetic characterisation of nAChRs. In 1985, @mishina1984 generated DNA constructs containing cDNA sequences encoding for the muscle type nAChR. These sequences were injected into the *Xenopus oocytes* [@mishina1984]. Upon application of acetylcholine, a current was recorded, suggesting successful cell surface expression. Single channel recordings revealed that in the presence of agonist, nAChR channel switches between active and inactive form. The active form comprises short-lived channel closing and opening [@mishina1986] and longer pauses in-between the receptor twitching. In addition, channel opening does not seem to be an all or nothing event. Instead, a channel exhibits multiple conductance states, one on which it is fully opened, named a full conductance state, and on in which the channel is partially opened, names sub-conductance state [@nagata1996; @nagata1998].  -->

<!-- Intracellulal recordings have shown that this is not due to the muscle cell itself, but rather the insensiticity of membrane receptors [@thesleft1955].  -->

<!-- $\alpha$-bungarotoxin ($\alpha$-bgtx), is a 74-amino acid long, 8 kDa proteins isolated from the venom of a snake *Bungarus multicinctus*. It binds with high affinity to the NMJ post-synaptic membranes [@lee1967] and blocks synaptic responses evoked by acetylcholine and other agonists [@chang1963] by blocking the access of an agoinist to the nAChR binging site [@@mishina1984].  -->

<!-- Behavioural analysis of Drosophila mutants in which cholinegic neurotransmission is diminished, provides evidence for the role of cholinergic neurotransmission in insects. Drosophila Ace null mutants are lethal: flies dye in the early development [@hall1976; @greenspan1980]. CAT mutants Lethal null mutation of drosophila choline acetyltransferase lethal flies dye during embryogenesis. some homozygus are viable but only at 18 deg c (temp sensitive) - some are temperature sensitive: viable at 18 deg due to the sufficient enzyme activity which is dimonished when flies placed in 30 deg. Upon sfift to high temp: characterisitc behaviour: loss of coordinated movement leading to paralysis and evenual death [@greenspan1980b] and disrupted cotrtnership behaviour. REDUCED MOTILITY IN vesicular acetylcholine transporter gene (Vacht) mutant [@kitamoto2000].  -->

<!-- It is effective against plant insect pests [], and used in organic farming in form of tobacco tea [@isman2006]  -->

<!-- Exposure of insects to lethal dose of nicotine results in symptoms characteristic for the nervous system intoxication [@chadwick1947] and include increased locomotory activity, followed by convulsions, twitching and eventual death [@mcindoo1943]. -->

#### Localisation of the cholinergic neurons in insects ####{#localisationininsects}

Enzymes, transporters and receptors present at the cholinergic synapse have been used as markers for detection of cholinergic neurons in insects. (1) Immunocytochemistry with monoclonal antibodies specific to ChAt and ACE, (2) *in-situ* hybridization using sequences complementary to the ChAT mRNA (3) colorimetric technique for detection of AChE activity [@karnovsky1964] (4) and reporter gene fused to the ChaT gene regulatory elements, outlined the presence of cholinergic pathways in *Drosophila* [@buchner1986; @gorczyca1987; @barber1989; @yasuyama1999], honeybee [@kreissl1989] and locust *Locusta migratoria* [@lutz1987; @geffard1985]. Based on these data, cholinergic neurons are in almost all regions of the brain and in the peripheral nervous system, namely the visual system and the antenna. They are also present in the thoracic, abdominal and the terminal abdominal ganglia involved in the regulation of movement of wings, abdomen and legs, as well as the regulation of the anal and reproductive muscles in insects [@smarandache-wellmann2016]. 

<!-- . In particular, the muchroom bodies associated with learning, formation of memory and the sensory processing [@heisenberg1998], dorsal lobe where mechanosensory and gustatory neurons project into from the anteanna -->

<!-- In peripheral nervous tissue, cholinergic neurons are the   -->
Cholinergic neurons have also been mapped using radiolabelled ligand, specific for nAChRs. $\alpha$-bungarotoxin ($\alpha$-bgtx), is a 74-amino acid long, 8 kDa proteins isolated from the venom of a snake *Bungarus multicinctus*. It binds with high affinity to nAChR [@lee1967] and blocks synaptic responses evoked by acetylcholine and other nAChR agonists [@chang1963]. Incubation of the honeybee brain with $\alpha$-bgtx led to staining in the optic lobes, antenna lobes, ocellar system and mushroom bodies [@scheidler1990]. This correlated with the staining in the central nervous system of *Drosophila* [@schmidt-nielsen1977], moth *Manduca sexta* [@hildebrand1979] and *cocroach* [@orr1990]. Incubation of $\alpha$-bgtx with the ganglia of the american *cocroach* [@sattelle1983] and cricket *Acheta domesticus* [@meyer1985] identified further regions where $\alpha$-bgtx binds with high affinity: the abdominal ganglion in the region rich in interneurons which make synaptic connections with the sensory afferent neurons [@daley1988], the abdominal ganglia and the thoracic ganglia [@sattelle1981]. Presence of nAChRs at the insect ganglia was confirmed using electrophysiological approaches [@sattelle1981; @bai1992].

Based on the distribution of cholinergic-synapse markers in the insect nervous system and the quantitative analysis of acetylcholine in the insect brain [@florey1963], it was concluded that acetylcholine is a major neurotransmitter in the nervous system of insects. In contrast to vertebrates [@brown1936; @bacq1937; @chang1963] and *C. elegans* [@richmond1999], acetylcholine in insects does not mediate muscle contraction at the NMJ, instead it is mainly involved in the sensory pathways and central information processing. The action of ACh in insects is exerted by nAChRs.

### Role of nAChRs in insects 
<!-- Lerning: [@kerkut1970] acetylcholinesterase inhibitor facilitated the ability of *cocroach* to learn to raise its leg out of the solution in response to electrical stimuli. Drugs: neostigmine and physostigmine which inhibit acetylcholinesterase [@carlyle1963]. -->

Biological role of nAChRs in insects was investigated in behavioural assays in response to nAChR agonists. Lethal doses of neonicotinoid imidacloprid induced complex symptoms in American *cocroach* and in honeybee [@sone1994; @elbart1997; @suchail2001]. The following order of events was noted: hyperexcitation as evident by excessive pacing, collapse and diminishing uncoordinated leg and abdomen movement followed by paralysis and eventual death. Lethal dose of insecticide nicotine [@david1953], a naturally occurring alkaloid found in the *Solanaceae* family of plants [@steppuhn2004], induced similar effects on bees [@mcindoo1943]. Distinct behavioural alterations can be induced by sub-lethal doses. Imidacloprid at < 4 nM inhibits feeding of *Myzus persicae*, which leads to their starvation [@nauen1995; @elbart1997]. In honeybees and bumblebees neonicotinoids impair on learning and memory, as well as reproduction (Section \@ref(sublethalbees)), highlighting an important role of nAChRs in insects biology. 

### Electrophysiological properties of insect nAChRs ###{#eletrophysinsectnachr}

The kinetic properties of insect nAChRs were investigated using neuronal preparations, where high density of nAChRs was found (Section \@ref(localisationininsects)). Acetylcholine and nicotine increased the rate of neuronal firing [@callec1973; @sattelle1976; @meyer1985; @kerkut1969; @sattelle1981; @bai1992] by depolarising post-synaptic neurons [@callec1973; @sattelle1976; @goldberg1999; @barbara2005; @brown2006; @palmer2013]. These effects were inhibited by nAChR antagonist $\alpha$-bgtx, suggesting effects of nicotine and acetylcholine were induced directly acting on nAChRs and that nAChRs are excitatory. Indeed, analysis of the agonist-evoked nAChR currents in the cultured honey bee neurons showed flux of mainly sodium and potassium but also calcium [@goldberg1999]. 

<!-- *cocroach* terminal abdominal ganglion extracellular post synaptic potentials in response to acetylcholine. In the presence of acetylcholinenesterase inhibitor acetylcholine at much lower doses 1 uM - 1 mM elicited Rapid and concentration dependent depolarisation excitation of the post-synapse and the gradual decline of the EPSP leading to their block [].  -->

<!-- were recorded intracellularlu t  -->

 <!-- recording of the interneuron activity nicotine - cholienrgic and evidence of nAChr expression from the biochem studies. In response to  blocked by bgtx [@meyer1985]  -->

 <!-- and was blocked by selective antagonist mecamylamine .  -->

#### Single channel kinetics

Single channel recordings showed that insect nAChRs exhibit complex kinetics, resembling those found in vertebrates [@colquhoun1985; @nagata1996; @nagata1998]. Using cholinergic neurons of the larva *Drosophila* CNS [@albert1993; @brown2006], and cultured neurons of *Musca domestica* (*M. domestica*) [@albert1993] it was shown that in response to nAChR agonists acetylcholine, nicotine, imidacloprid and clothianidin, the channel switches between an active and inactive form, with the active form interrupted by the short-lived channel closing bursts. Temporal characterisation of these events revealed that the frequency of channel opening and the duration of opening differs depending on the agonist applied and the neuronal preparation. However, typically the receptor remains opened for ~ 1.5 ms; this is interrupted by channel closing bursts of ~ 20 $\mu$s which occur at a frequency of 1-2 closures/opening burst [@albert1993]. 

Channel opening is not an all or nothing event. Instead, a channel typically exhibits two conductance states, one on which it is fully opened, named a full conductance state (i.e. the active form), and one in which the channel is partially opened, named the sub-conductance state. Although the conductance rates from various insect preparations are similar, the ratio between the two as well as their fine structure varies depending on the concentration, the agonist used and and the neuronal preparation [@albert1993; @brown2006].

#### Desensitisation of insect nAChRs

Exposure of insect neuronal preparations to high concentrations of agonists has a secondary effect. Following rapid depolarisation, the current slowly decreased until it it abolished completely due to nAChR desensitisation [@goldberg1999]. Desensitisation is a period after agonist removal, whereby subsequent depolarisation cannot be elicited by agonist [@goldberg1999]. The time taken for desensitisation varies between hundreds of mseconds [@goldberg1999] to tens of seconds [@salgado2004] in insects. In vertebrates, there are receptors which desensitise in $\mu$seconds [@bouzat2008]. Although the process of receptor desensitisation is typically reversible [@goldberg1999; @salgado2004], full recovery may not occur or may be slower if the receptors are exposed to large doses of agonist for a prolonged time [@katz1957].

<!-- Immunocytochemistry using monoclonal antibodies specifie -->
<!-- In the brain, cholinergic neurons can be found in almost all parts of the brain and the optical lobe. In particular, mushroom bodies,  -->

<!-- thoracic ganglia : afferent sensory nurons  -->

<!-- In the peripheral primary sensory nerouns of the compound eye and the anntena.  -->

<!-- Afferent sensory neurons  -->


<!-- interneurones of the thoracic ganglia  -->




<!-- Cholinergic neurons in the cortical regions of almost all regions of the insect brain -->

<!-- Immunocytochemistry  -->

<!-- Biochemical techniques using monoclonal antibodies specific agains  -->

<!-- Large quantities of achetylcholine isolated from the insect brain preparations  -->

<!-- also "immunoreactivity first, in -->
<!-- regions of neuropile previously shown by backfilling -->
<!-- with cobalt to contain terminals from sensory neurones. Some roots of peripheral nerves also bind the antibody. Secondly, a small number of neurone cell bodies in the brain and thoracic ganglia are immunoreactive and we assume these belong to interneurones" and " first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the -->
<!-- ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies" [@lutz1987].  -->

<!-- immunoreactivity in almost all brain regions of *D. melanogaster* " apart from -->
<!-- the lobes and the peduncle of the mushroom body and -->
<!-- most of the first visual neuropile (lamina)" [@buchner1986]. -->

<!-- in-situ hybridization: [@barber1989] "Substantial amounts of cRNA probe for ChAT mRNA -->
<!-- were observed associated with cells of all cortical regions of -->
<!-- the optic lobe and brain. Neuropil regions of the -->
<!-- brain displayed insignificant amounts of hybridization with -->
<!-- the experimental probe and specimens incubated with the control probe did not exhibit significant -->
<!-- hybridization in either cortical or neuropil areas . -->
<!-- In peripheral nervous tissue, there was substantial labeling over primary sensory neurons in the antenna, and significant amounts of probe were associated with the -->
<!-- retinular cell layer of the compound eye ". the compound eye is the visual system of the fly. -->

<!-- AND " histochemical detection of reporter gene expression " using x-gal : [@yasuyama1999]. -->



<!-- " cholinergic primary sensory neurons in the Drosophila antennal system"  -->

<!-- aceti-cholinesterase :  colorimetric determination of acetylcholinesterase activity with -->

<!-- Histochemistry of AChE with colorimetric assay using isothiocholine:  -->
<!-- "the optic lobes, fibers connecting the two brain hemispheres, and fiber tracts -->
<!-- as well as soma clusters within the protocerebrum. The calycal input regions of -->
<!-- the mushroom bodies were labelled, whereas the intrinsic Kenyon cells -->
<!-- showed no staining. Although the antennal afferents projecting into the dorsal -->
<!-- lobe showed strong AChE activity, projections into the antennal lobe showed -->
<!-- rather weak staining." [@kreissl1989] the same paper shows a-bungarotoxin (a-BTX) binding sites and AChR monoclonal antibodies with a good overlap in "d immunoreactivity -->
<!-- in neuropiles, tracts, somata, and the antennal nerve. The immunoreactivity -->
<!-- of the optic lobes coincided with the banding pattern of the AChE staining. A -->
<!-- particularly striking overlap of AChR immunoreactivity and AChE staining -->
<!-- was found in the lip neuropile of the mushroom bodies" all in the honeybee -->



<!-- check this paper to see evidence for cholinergic neurons "between the -->
<!-- sensory neurons and interneurons of the cockroach cercal sensory system " paper: " The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine" -->


<!-- Its action is mediated predominately by nAChRs, which are the main cholinergic receptor type in their central nervous system [@breer1987].  -->

<!-- The presence of nAChR in various brain regions has been detected using biochemical and electrophysiological techniques on neuronal preparations extracted from the Fruit fly *Drosophila melanogaster*, honey bee *Apis mellifera* and .  -->


<!-- nAChRs have been found to be expressed in the regions  -->

<!-- , namely the muschroom bodies [@kreissl1989; @gu2006; @oleskevich1999]. -->



<!-- In contrast to mammals and invertebrates (Table \@ref(tab:chlinergic-nts)), insects do not express nAChRs at the neuromuscular junction.  -->

<!-- ##### Electrophysiology  -->
<!-- Evidence from the application of nAChR:  -->

<!-- nicotine:  -->
### Structural basis of major conformation states of nAChRs

Nicotinic acetylcholine receptors have three basic conformation states: the closed, the open and the desensitised state [@katz1957; @monod1965]. Structural features of the closed state channel are described in Section \@ref(structure) and \@ref(modelodnachbinding)). Briefly, nAChR is a pentameric assembly of receptor subunits. Each subunits contains 4 transmembrane helices (M1-M4) [@noda1982; @noda1983], an N-terminal helix and 10 $\beta$sheets [@brejc2001; @dellisanti2007; @li2011] and a large C-terminal domain [@unwin1995; @dellisanti2007; @li2011]. The N-terminal domain contains an agonist binding site formed by the loop contributions from the adjacent subunits [@brejc2001]. One of the key features of the closed-channel is the presence of leucine residues originating from the pore-lining M2 helix, which project inwards [@unwin1995]. These residues form a gate which occludes the passage of ions of closed nAChR. High resolution structures of AChBP [@bourne2005; @hansen2005] and human $\alpha7$-AChBP chimera [@li2011] highlighted the structural differences between the open (agonist-bound) and the closed states. In the agonist-bound structures, the aromatic residues in C loop form a cap above the agonist, suggesting that ligand binding leads to movement of the C-loop which folds over the agonist binding site, burying the ligand inside the protein and reducing the dissociation on/off rates. In addition, loop A moves towards the loop B, whereas loop F moves towards the agonist. These local changes propagate the rearrangement of the outer $\beta$ sheet which rotates towards the centre of the pentamer and lead to structural changes at the level of the channel, leading to its opening.

Crystal structures of bacterial pentameric ligand gated ion channels shed light on the possible mechanism of channel opening. Although these channels are not members of the Cys-loop family due to the absence of N-terminal disulphide bond and a large cytoplasmic loop between M3-M4 transmembrane (TM) helices, they share common topology with nAChRs. Comparison of closed *Erwinia chrysanthemi* ligand gated ion channels (ELIC) [@hilf2008] to opened *Gloeobacter violaceus* ligand gated ion channels (GLIC) [@hilf2009], showed that in the open state pore-lining helices are tilted inwards, which leads to opening of the gate. An alternative hypothesis of channel opening was derived based on the cryo EM of the mammalian muscle nAChR in closed and open state [@unwin1995]. These structures suggest that binding of the agonist leads to rotation of 5 M2 helices. As they move, the distance between them increases, and so the ion conductivity pathway becomes wider, the gate opens, thus ions flow. More recently a higher resolution structure of muscle type nAChR has been derived [@unwin2012], suggesting that in the open state, TM helices not only rotate, but also bend towards the centre of the pore. Twisting and tilting of inner helices were also observed in the crystal structures of other representative of Cys-loop receptors, namely glycine receptors [@du2015] and glutamine-gated chloride (GluCl) channel [@althoff2014])

In 2016, the crystal structure of the human $\alpha4$/$\beta2$ receptor in desensitized state [@morales-perez2016] was derived. This was compared to the structures of open glycine [@du2015], closed GluCl [@althoff2014] and desensitized GABA [@miller2014]. Differences at the interface of the extracellular domain (ECD) and TM regions were noted, which arise as a result of the rotation motion at the level of the receptor. The structural rearrangements lead to the occlusion of the ion channel, reducing conduction [@monod1965] and tightening of the ligand binding site leading to an increase in ligand affinity to the desensitised receptor [@monod1965].

### Neonicotinoids act as nAChR agonists ### {#neonicstarget}

#### Mutations in nAChRs give rise to neonicotinoid-resistance ####{#resgenevidence}

Several lines of evidence suggest that nAChR are the principal site of action of neonicotinoids. Genetic analysis of the neonicotinoid-resistant strains of insects showed that resistance arises as a consequence of mutations in nAChR subunits. Field isolates of peach aphid *M. persicae* [@bass2011], the cotton aphid *A. gossypii* [@hirata2015; @hirata2017] and the Colorado potato beetle *L. decemlineata* [@szendrei2012], as well as lab-isolates of brown planthopper, *N. lugens* [@liu2005], fruit fly *D. melanogaster* [@perry2008] with decreased sensitivity to neonicotinoids have been identified. Behavioural analysis shows that their sensitivity is up to 1500-fold lower in comparison to the reference strains, as shown by the shift in the LD~50~. Analysis of the DNA of the resistant strains identified mutations in nAChR subunit coding sequence [@bass2011; @perry2008; @hirata2015]. However, mutations in other genes can also give rise to resistance [@zewen2003; @Benting2004; @bass2015].

#### Neonicotinoids evoke nAChR-like current in insect neuronal preparations ####{#electrophysevidence}

Neonicotinoids induce nAChR-like current in insect neuronal preparations, which reassembles that induced by nAChR agonist nicotine (Section \@ref(eletrophysinsectnachr)). @sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *P. americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained increase in the rate of neuronal firing for over 2 minutes. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the *L. decemlineata* [@tan2008] and on the abdominal ganglion of *P. americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984].

 <!-- This phenomenon is due to the distinct conformation of nAChRs, at in which they are not capable of ion conduction [@nemecz2016].  -->

<!-- In the continual presence of agonist, the membrane potential gradually repolarises until it reaches the resting state, hovewer the next depolarisation will not occur, until the agonist are washed off and the receptor recovers from desensitisation [@katz1957]. -->

@sattelle1989 used isolated *cocroach* neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated *cocroach* thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured *cocroach* [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons.

Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC~50~ values (concentrations at which the half of the maximum current was observed) were in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(potentpests)). Neonicotinoid-induced currents were reminiscent of those induced by acetylcholine and nicotine, and were prevented by the application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine), suggesting neonicotinoid-induced currents are due to the activation of nicotinic receptors.

### Mode of action of neonicotinoids ###{#moaneonicsinsects}

Although neonicotinoids typically acts as agonists, they can have diverse mode of action. The currents produced by neonicotinoids and ACh on cultured or isolated insect neuronal preparation were compared. Neonicotinoids evoking current lower than that evoked by ACh were classed as partial agonists, those eliciting similar response were classed as true agonists, whereas those more efficacious than ACh, super-agonists. Thiacloprid and imidacloprid were shown to be partial agonists, nitenpyram, clothianidin, acetamiprid and dinotefuran were true agonists, whereas thiamethoxam had no effect on the isolated American cockroach thoracic ganglion neurons [@tan2007]. This differs from the mode of action of neonicotinoids on cultured terminal abdominal ganglion neurons of this insect. Currents produced by all neonicotinoids tested was lower than that evoked by ACh [@ihara2006], suggesting they are all partial agonists on these cells. The mode of action of neonicotinoids on the fruit fly [@brown2006] and honey bee neurons [@palmer2013] differs still, implying the presence of distinct nAChRs in different insect species and neuronal preparations. 

#### Neonicotinoids bind with high affinity to insect nAChRs ####{#ligbinding}

<!-- @tomizawa1996 developed neonicotinoid agarose affinity column to isolate proteins with high binding affinity to neonicotinoids. Using Drosophila and Musca head membrane preparations, he identified three nAChR subunits as potential neonicotinoid-targets.  -->

Neonicotinoids bind to insect nAChRs with high affinity, as shown in the saturation ligand binding studies. In the saturation binding experiment, various concentration of the labelled ligand is added to the preparation and the concentration of the ligand at the equilibrium is determined. This is then used to derive dissociation constant, Kd, to define the binding strength of neonicoinoids to insect nAChRs. In the whole membrane preparations of the housefly and aphid, the Kd of imidacloprid and thiamethoxam were in the low nM range [@liu1993; @wellmann2004; @liu2005] suggesting high affinity interaction. Interestingly, two binding affinities have been derived from the imidacloprid study in the brown planthopper and pea aphid [@wellmann2004; @taillebois2014] suggesting the presence of at least two imidacloprid binding sites in these animals.

```{r potencyintact, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
 library(dplyr)

 potencyintact <- data.frame(
   Compound = c("Imidacloprid", "Imidacloprid", "Imidacloprid", "Imidacloprid", "", "Imidacloprid", "", "Thiamethoxam"),
   Species = c("\\textit{Musca domestica}", "\\textit{Aphis craccivora}", "\\textit{Myzus persicae}", "\\textit{Nilaparvata lugens}", "", "\\textit{Acyrthosiphon pisum}", "", "\\textit{Myzus persicae}"),
   Common = c("housefly", "cowpea aphid", "green peach aphid", "brown plantohopper", "", "pea aphid", "", "green peach aphid"),
   Kd = c("1.2", "12.3", "4.1", "<0.01", "1.5", "0.008", "0.002", "15.4"),
   References = c("Liu et al. 1993", "Wellmann et al. 2004", "", "Liu et al. 2005", "", "Taillebois et al. 2014", "", "Wellmann et al. 2004"))
   
 potencyintact %>%
    mutate_all(linebreak) %>% 
 kable("latex", align = "l", booktabs = TRUE, escape = F,
       col.names = linebreak(c("Compound", "Species", "Common\nname", "Kd\n(nM)", "References")), 
       caption = 'Binding affinity of neonicotinoids') %>%
  kable_styling(position = "left", full_width = FALSE, latex_options = "hold_position") %>%
                add_footnote(notation = "none", "Binding affinity measured in the whole membrane preparation, with the exception of housefly experiment where head membranes were used.", 
                             threeparttable = T)
```

In addition to the saturation studies, the competitive ligand binding studies were carried out. In the the competitive ligand binding studies, biological preparation is incubated with radiolabelled ligand. The ability of various concentrations of unlabeled ligand is measured to define its equilibrium inhibition constant (Ki). This method informs both on the affinity and on the interactions between ligands. Various concentrations of neonicotinoid prototype isothiaocynate were incubated with the homogenate of *Drosophila* and a homogenate of the abdominal nerve cords of *P. americana* before the exposure to radiolabelled nAChR antagonist $\alpha$-bgtx [@gepner1978]. Isothiaocynate inhibited binding of $\alpha$-bgtx in the concentration dependent manner [@gepner1978], suggesting the two compounds share the binding site. Similarly, imidacloprid has been shown to displace $\alpha$-bgtx from brain membrane preparations from honey bee *A. mellifera* [@tomizawa1992; @tomizawa1993], *Drosophila* [@zhang2004], house fly *M. domestica* and isolated *cockroach* nerve cords [@bai1991]. The binding affinity of neonicotinoid-related compounds was compared to the insecticidal activity; the correlative relationship between the two was found [@kagabu2002; @liu2005], providing further evidence that neonicotinoids act by targeting nAChRs. 

#### High affinity of neonicotinoids to heterologously expressed insect-chimera receptors ####{#chimerareceptors}

Due to the difficulties in the heterologous expression of native insect receptors (Section \@ref(expressionfail)), the binding affinity of neonicotinoids to isolated, native receptors is largely unknown. However, binding studies on hybrid receptors consisting of insect $\alpha$-subunit and vertebrate $\beta$ subunit, were carried out.

<!-- determined. $\beta2$ from rat and chicken has been shown to enable recombinant expression of several insect $\alpha$ subunits in cell lines.  -->

Mammalian $\alpha4$/$\beta2$ receptor expresses well in *Xenopus* oocytes [@cooper1991] and cell lines [@lansdell2000] and it has low affinity to imidacloprid (Kd >1000 $\mu$M) [@lansdell2000].  Chimera of rat $\beta2$ and $\alpha$ subunits from the fruit fly [@lansdell2000], aphid *M. Persicae* [@huang1999], planthopper *N. lugens* [@liu2009], cat flea *Ctenocephalides felis* [@bass2006] and sheep blowfly *Lucilia cuprina* [@dederer2011] have been generated. It needs to be noted that the potency of neonicotinoids on these receptors is not reported, suggesting these receptors are not functional. However, their pharmacological profiles have been determined using saturation ligand binding studies [@hulme2010] (Table \@ref(tab:bindignrecombinant)). 

The affinity of neonicotinoids to insect-chimera rectors varies, depending on the identity of the $\alpha$ subunit. Imidacloprid did not bind to Mp$\alpha1$/rat$\beta2$ receptor, whereas its Kd at Mp$\alpha2$ and Mp$\alpha3$-containing receptor was 3 and 2.8 nM, respectively [@huang1999]. Four to five fold-difference between the most and least susceptible fruit fly and cat flee receptor assemblies were also identified [@lansdell2000; @bass2006]

Imidacloprid exhibits the highest affinity against target pest *M. Persicae* with the lowest reported Kd of 2.8 nM on $\alpha3$/$\beta2$ receptor [@huang1999]. It binds less tightly to the non-target insect, the fruit fly nAChRs; the Kd values range from 8.4 to 34.9 nM [@lansdell2000].

```{r bindignrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
 library(dplyr)

footnotez <- ("Receptors were expressed in insect S2 cell line")
footnotey <- ("Rn = Rattus norvegicus (rat), Dm = Drosophila melanogaster (fruit fly), Mp = Myzus persicae (aphid), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), N/B = no binding,")

 bindingrecombinant <- data.frame(
   Receptor = c("Rn$\\alpha4\\beta2$", "Dm$\\alpha1$/Rn$\\beta2$", "Dm$\\alpha2$/Rn$\\beta2$", "Dm$\\alpha3$/Rn$\\beta2$",  "Mp$\\alpha1$/Rn$\\beta2$", "Mp$\\alpha2$/Rn$\\beta2$", "Mp$\\alpha3$/Rn$\\beta2$", "Mp$\\alpha4$/Rn$\\beta2$", "Nl$\\alpha1$/Rn$\\beta2$", "Cf$\\alpha1$/Dm$\\alpha2$/Rn$\\beta2$", "Cf$\\alpha3$/Dm$\\alpha2$/Rn$\\beta2$"),
   Kd= c(">1000", "34.9", "20", "8.4", "N/B", "3", "2.8", "N/B", "24.3", "141", "28.7"),
   References = c("Lansdell and Millar, 2000", "", "", "", "Huang et al., 1999", "", "", "", "Liu et al., 2005", "Bass et al. 2006", ""))
   
 bindingrecombinant %>%
    mutate_all(linebreak) %>% 
 kable("latex", align = "c", booktabs = TRUE, escape = F,
       col.names = linebreak(c("Receptor", "Kd\n(nM)", "Rerefence")), 
       caption = 'Binding affinity of imidacloprid to recombinant insect-hybrid receptors') %>%
   kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
    add_footnote(notation = "none", c(footnotez, footnotey),
            threeparttable = T)
```

#### High potency of neonicotinoids on heterologously expressed insect-mammalian hybrid receptors 

The potency of neonicotinoids on insect-mammal hybrid nAChRs have been also determined using cyanoamidines clothianidin and imidacloprid, nitroguanidines thiacloprid and acetamiprid and nitromethylene nitenpyram.

Dose-dependent depolarising current was recorded from cells expressing insect-hybrid nAchRs in responses to all tested neonicotinoids. The potency of neonicotinoids varied, as indicated by the EC~50~ values between 0.04 and 45.8 $\mu$M, however it is generally in the region of 1 $\mu$M. 

The rank order of potency of cyanoamidines, nitroguanidine and nitromethylene differs, depending on the receptor identity. For example, in imidacloprid and clothianidin are the most potent on the fruit fly $\alpha1$ containing receptors [@dederer2011], whereas planthopper $\alpha3\alpha8$ hybrid, thiacloprid is the most potent [@yixi2009]. Nitenpyram has consistently the highest EC~50~. 

```{r potencyrecombinant, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
 library(dplyr)

footnotew <- ("Receptors were expressed in Xenopus oocytes")
footnotex <- ("Rn = Rattus norvegicus (rat), Gg = Gallus gallus (chicken), Dm = *Drosophila melanogaster (fruit fly), Nl = Nilaparvata lugens (planthopper), Cf = Ctenocephalides felis (cat flea)), Lc = Lucilia cuprina (sheep blowfly)")

 potencyrecombinant <- data.frame(
   Receptor = c("Nl$\\alpha1$/Rn$\\beta2$", "Nl$\\alpha2$/Rn$\\beta2$", "Nl$\\alpha3$/Rn$\\beta2$", "Nl$\\alpha3\\alpha8$/Rn$\\beta2$", "", "", "", "Dm$\\alpha1$/Gg$\\beta2$", "", "", "", "Dm$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha1$/Gg$\\beta2$", "", "", "", "Cf$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha4$/Gg$\\beta2$", "", "", ""),
   Compound = c("Imidacloprid", "Imidacloprid", "Imidaclorprid", "Imidacloprid", "Clothianidin", "Thiacloprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram"),
   EC50 = c("61", "870", "350", "3.2", "5.1", "2.8", "5.6", "0.04", "0.34", "0.23", "0.4", "0.84", "5.4", "2", "35.4", "0.02", "0.15", "0.11", "0.63", "1.31", "1.65", "2.63", "24.4", "13.8", "21.3", "9.4", "45.8"),
   References = c("Liu et al. 2009", "", "", "Yixi et al. 2009", "", "", "", "Dederer et al. 2011", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", ""))
   
 potencyrecombinant %>%
    mutate_all(linebreak) %>% 
 kable("latex", align = "c", booktabs = TRUE, escape = F,
       col.names = linebreak(c("Receptor", "Compound", "EC50\n($\\mu$M)", "Rerefence")), 
       caption = 'The potency of neonicotinoids on recombinantly expressed insect hybrid nAChRs.') %>%
  kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
    add_footnote(notation = "none", c(footnotew, footnotex),
            threeparttable = T)
```

<!-- Based on the expression of several potential assemblies have been identified. -->

<!-- Subunits with various degree of sensitivity to neonicotinoids, suggesting some and not all receptors confer neonicotinoid sensitivity.  -->

<!--  cultured Drosophila CNS cholinergic neurons, whereas imidacloprid is a partial agonist there .  -->

<!-- Clothianidin and imidacloprid are partial and full, agonist, respectively on the isolated Kenyon cells of the honeybee . 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 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]. -->

<!-- Note that neonicotinoids cause depolarizing block by desensitising receptors [@palmer2012]. -->


<!-- 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 imidacloprid 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)). -->

<!-- In addition, nAChR-like current was evoked in Xenopus oocytes transfected with membrane preparations of pea aphid *Acyrthosiphon pisum* in response to clothianidin, acetylcholine and nicotine [@crespin2016].  -->


<!-- nAChRs are expressed pre-, post- synaptically at the cell body, as determined mainly with electrophysiological approaches, whereby upon application of classical agonists (Section \@ref(pharma-generalintro)), nAChR-like currents were elicited from various parts of neuronal preparations. The agonist-induced currents were blocked with nAChR antagonists. The presence of nAChRs in the Fruit fly *Drosophila melanogaster* (refereed to as Drosophila, in short) Kenyon cells was shown by recording spontaneous and nicotine or acetylcholine-evoked post-synaptic currents of neurons. 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]. -->

```{r chlinergic-nts, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)

footnote1 <- "NT = neurotransmitter, NMJ = neuromuscular junction"
footnote2 <- "References: 1 = Chen and Patrick 1997, 2 = Araujo et al. 1988, 3 = Couturier et al. 1990; Cooper et al. 1991, 4 = Lee et al. 1967; 5 = Brown et al. 1936, 6 = Mishina et al. 1986, 7 = Zirger et al. 2003, 8 = Mongeon et al. 2011, 9 = Lewis et al. 1987, 10 = Treinin et al. 1998, 11 = Richmond and Jorgensen 1999; 12 = Boulin et al. 2008, 13 = Touroutine et al. 2005, 14 = McKay et al. 2004"


library(kableExtra)
 library(dplyr)
 cholnts <- data.frame(
   Species = c("Mouse", "\\textit{D. melanogaster}", "\\textit{D. rerio}", "\\textit{C. elegans}", "\\textit{A. mellifera}"),
   Localisation = c("Nervous system\nNMJ", "Nervous system", "Nervous system\nNMJ", "Nervous system\nNMJ", "Nervous system"),
   Function = c("NT release modulation\nMuscle contraction", "Major NT", "NT release modulator\nMuscle contraction", "Major NT\nMuscle contraction", "Major NT"),
  Major = c("$\\alpha4\\beta2$ and $\\alpha7$\n$\\alpha1\\beta1\\epsilon\\delta$", "?", "$\\alpha4\\beta2$ and $\\alpha7$\n$\\alpha1\\beta1\\epsilon\\delta$", "DES-2/DEG-3\nL-, N-type and EAT-2", "?"),
   Ref = c("1-3\n4-6", "in-text", "7\n8", "9, 10\n11-14", "in-text"))
 
 cholnts %>%
   mutate_all(linebreak) %>%
 kable("latex", align = "l", booktabs = TRUE, escape = F,
       col.names = linebreak(c("Species", "Localisation\nof nAChRs", "Function\nof nAChRs", "Major\nreceptor types", "Ref")),
       caption = 'Nicotinic acetylcholine receptors in insects, nematodes, mammals and fish',
       ) %>%
   kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
   add_footnote(notation = "none", c(footnote1, footnote2),
            threeparttable = T)
```

\newpage

### nAChR subunits in insects ###{#expressionfail}
The eletrophysiological and ligand binding studies on neuronal preparations and hybrid receptors provides evidence that nAChR are molecular targets of neonicotinoids. 
nAChR are assemblies of 5 different or identical receptor subunits (Section \@ref(structure)). Each subunit is encoded by a separate gene and is classified as either $\alpha$ or non-$\alpha$, depending on the primary amino acid sequence, whereby $\alpha$ subunits contain a disulphide bond formed between the adjacent cysteines in the ligand binding domain (Figure \@ref(fig:structure-nachr-label)). Genome sequencing projects enabled identification of nAChR subunit families in several insect species. Fruit fly and model organism *D. melanogaster* has 10 subunits, 7 of which are $\alpha$ ($\alpha1-7$) and 3 are $\beta$ ($\beta1-3$) [@adams2000a; @sattelle2005]. There are 11 subunits in the beneficial insect honeybee *A. mellifera* ($\alpha1-9$, $\beta1-2$) [@jones2006a; @consortium2006], 12 subunits in the pest red flour beetle *Tribolium castaneum* ($\alpha1-11$,, $\beta1$) [@consortium2008] and 8 in the Pea Aphid, *Acyrthosiphon pisum* ($\alpha1-6$, $\beta1-2$) [@yi-peng2013; @Consortium2010]. With the aid of molecular cloning techniques, equivalent subunits have been identified in many other insects, including cat flea *C. felis* [@bass2006] and green peach aphid *M. persicae* [@huang2000]. Amino acid sequence alignment of equivalent subunits revealed that they are highly conserved, with sequence identity typically greater than 60% [@jones2010].

Insect nAChR gene families are among the least diverse when compared to other animal phyla. Mammals express 17 subunits: $\alpha1-10$, $\beta1-4$, $\delta$, $\gamma$ and $\epsilon$ [@millar2009] and there are 29 subunits in the representative of the phylum *Nematoda, C. elegans* [@jones2007a].

### Difficulties in heterologous expression of insect nAChRs ###{#difficuktiesinexpressioninsectreceptro-intro}
<!-- There are different receptor types in insects. -->

<!-- https://radar.brookes.ac.uk/radar/file/c59cbdb5-d171-49e0-b0e4-101c261c72ed/1/fulltext.pdf -->

To identify which subunits assemble to form functional receptors, recombinant expression techniques were used. Recombinant expression is a technique by which receptor stoichiometry and function can be studied in a heterologous system. cDNA is injected into the *Xenopus* oocytes, or used to transfect insect or mammalian cell lines. Using internal cellular machinery, it is transcribed, translated and processed to the surface of the cell. Should a protein form, cell-surface expression can be detected using biochemical approaches (such as ligand binding studies), whereas function can be studied by means of electrical recordings. These approaches were utilised to identify the major receptor assemblies in mammals, nematode and fish (Table \@ref(tab:chlinergic-nts)). 

<!-- To determine which insect nAChR subunits come together to form a functional receptor, various nAChR subunits were injected into the expression systems (*Xenopus oocytes* or cell lines) to determine whether cell surface expression and receptor function can be obtained.  -->

To determine which insect subunits form functional nAChRs, @lansdell2012 transfected cultured insect cells with over 70 *Drosophila melanogaster* nAChR subunit cDNAs either singularly or in combinations. No cell surface was achieved, as shown by the radiolabelled ligand binding studies. Difficulties in expression of *Drosophila* subunits were also encountered in *Xenopus* oocytes [@lansdell2012] and mammalian cell lines [@lansdell1997]. The attempts to express receptors from other species were also largely unsuccessful. No ligand binding and/or agonist evoked currents were detected from cells transfected with genes en
coding for the nAChR subunits of brown planthopper [@liu2005; @liu2009; @yixi2009], cat flea [@bass2006], aphid *M. persicae* [@huang2000] and brown dog tick *Rhipicephalus sanguineus* [@lees2014]. Homomeric *Locust* $\alpha1$ [@marshall1990], *M. Persicae* $\alpha1$ and *M. Persicae* $\alpha2$ [@sgard1998] produced receptors with nAChR-like pharmacological and electrophysiological characteristics, however the channel-generated currents were of low amplitude, and the expression was inconsistent. 

##### Importance of chaperon proteins in heterologous expression of nAChRs ###{#ric3insect}

Difficulties in recombinant receptor expression highlight the complexity of receptor formation. Assembly and oligomerisation are critical steps in the receptor maturation [@brodsky1999] which are poorly understood in insects. Co-expression of mammalian and *C. elegans* nAChRs with chaperon proteins shed light on the critical role of endoplasmic reticulum (ER) and Golgi resident proteins in receptor maturation. @boulin2008 demonstrated that three chaperon proteins are necessary for the expression of *C. elegans* muscle-type receptors in *Xenopus* oocytes: UNC-50, UNC-74 and RIC-3 (described in more details in Sections \@ref(ric-3celegans); \@ref(unc50) and \@ref(unc74)); ligand binding and agonist-evoked currents were abolished upon exclusion of any of the three proteins. Resistant to inhibitors of cholinesterase-3 (RIC-3) protein also improves the cell surface expression of the second type of the BWM *C. elegans* receptor [@ballivet1996] and the neuron-type *C. elegans* receptor in *Xenopus* oocytes [@halevi2002]. It also plays a role in the maturation of human receptor in *Xenopus* oocytes and cell lines (Section \@ref(ric-3nacho)). More recently, RIC-3 has been shown to influence folding and maturation of insect nAChRs. Co-expression of Dm$\alpha2$-containing and Dm$\alpha5$/$\alpha7$ receptors with RIC-3 improved [@lansdell2008], and in some instances enabled expression in otherwise non-permissible systems [@lansdell2012]. Up to 3.5-fold increase in specific binding of radiolabelled antagonist was noted in insect cells co-transfected with RIC-3, suggesting the presence of greater number of folded receptors on the cell surface [@lansdell2008]. Expressed receptors have been also shown to be functional: in *Xenopus* oocytes, ionic currents were detected in response to acetylcholine [@lansdell2012]. 

Inability to heterologous express insect nAChRs hinders their pharmacological characterisation and identification of subunits important in conferring the agricultural role of neonicotinoids. Development of a platform in which the heterologous nAChRs from pest-insects and non-target organisms could be expressed, would enable pharmacological characterisation of these proteins to better define the mode of action and selective toxicity of neonicotinoids. It would also open the door to screening of novel insecticides, to combat emerging and spreading neonicotinoid-resistance (Section \@ref(resgenevidence)) and @charaabi2018).

<!-- <!-- RIC-3 enabled identification of potential insect nAChRs. Co expression of Drosophila $\alpha5$ and $\alpha7$ either singlularly or in combonation, gave rise to cell-surface expression, as shown by radiolabelled ligand binding [@lansdell] and heteromeric $\alpha5$/$\alpha7$. The identity of other insect receptors is unknown. -->

<!-- (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") -->
<!-- ``` -->


<!-- clothiandidin on the abdominal ganglion [@thany2009] and imidacloprid on the  Colorado potato beetle (CPB), .  -->

<!-- Imi IC5 0 = 0.4 mM EC50 = 18.3 nM these vaues are from @tan2007. note he also look at the resistant strain -->

<!-- single-electrode voltage clamp @tan2007 (intracellular) depolarisation and see for comparison of activity between all neonics  -->

<!-- @thany2009 also did patch clamp inward current  -->

<!-- @ihara2006 whole cell patch clamp inward current  -->

<!-- Mannitol-gap recordings - must read this was used @buchingham1997, @tan2007 - extracellular recording schematic in tan2007 -->

<!-- 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 assemblies [@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. -->

<!-- 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). -->

<!-- Receptor assembly and  -->

<!-- The role of RIC-3 (resistant to inhibitors of cholinesterase-3) protein in an ER residing proteinin receptor maturation was first identified by @@halevi2002, who demonstrated that .  Heterogous expression of *C. elegans*  however, upon co-expression of RIC-3 proteins, both receptor current and ligand binding were detected [@holevi2002].  -->


<!-- The stoichiometry of these receptors in unknown, however there are some potential assemblies. For example Drosophila receptors contain $\alpha4\beta3$, $\alpha1\alpha2\beta2$ and $\beta1\beta2$ subunits [@chamaon2002], 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. -->


<!-- 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]. -->

<!-- "connections between afferents sensory neurons with interneurons or with motoneurons in several insects such as the cockroach" -->

<!-- ### Recombinant receptors  -->

<!-- Recombinant insect nAChR are notoriously difficult to express. Several interventions have been tested including expression of hybrid receptor in which insect subunits have been co-expressed with mammalian ones. It need to be noted that this method has several limitations. First, hybrid receptors are not biologically relevant, thus conclusions from these studies should be drawn with caution. Second, some some of the expressed receptors may be folded, but not functional. Lastly, this method enabled expression of only a handful of receptors, thus most remained uncharacterised. This hinders their pharmacological characterisation and identification of subunits important in conferring the agricultural role of neonicotinoids. Heterologous expression of nAChR from insects and other species would allow for the characterisation of the interactions of these proteins with neonicotinoids to better define their mode of action and selective toxicity. Development of the platform in which the heterologous expression of insect nAChRs could be achieved, would open the door to screening of novel insecticides, to combat emerging and spreading neonicotinoid-resistance (Section \@ref(resgenevidence)) and @charaabi2018). In addition, by expressing nAChRs from pest and other species identification of compounds with no adverse effects on beneficial insects and other biologically important species may be achieved. Model organism *C. elegans* is a system in which the mode of action and the selective toxicity can be studied.  -->

## *C. elegans* as a model system for expression of nAChRs

As indicated, the expression of insect receptors is limited due to difficulties in heterologous expression in *Xenopus* oocytes or cell lines. This suggests that these systems do not offer appropriate cellular environment for receptor maturation. Model organism *C. elegans* is an alternative model in which heterologous receptor expression can be achieved [@crisford2011; @salom2012; @sloan2015]. 

*C. elegans* is a transparent non-parasitic nematode, inhabiting temperate soil environments. This worm was first described as a new species in 1900 [@maupas1900] and named *Caenorhabditis elegans* Greek *caeno* meaning recent, *rhabditis* meaning rod-like and Latin *elegans* meaning elegant. The natural isolate of this species was extracted from the compost heap in Bristol by Sydney Brenner in 1960's and named N2. Since, *C. elegans* has become a valued lab tool and a model organism due to ethical, economical and biological reasons. In contrast to vertebral organisms, *C. elegans* is not protected under most animal research legislation. The cost of use is low, due to the cost of purchase (~$6/strain), maintenance, fast life cycle and high fertility of these animals. *C. elegans* is also the first multicellular organism to have the whole genome sequenced [@consortium1998]. In addition, every single neuron of its nervous system was 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 of *C. elegans* ##{#genbiology}

*C. elegans* exists as a male and a hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9% of worms are hermaphrodites, which self-fertilise their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15$^\circ$C, it takes 5.5 days from egg-fertilisation to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25$^\circ$C, respectively (Figure \@ref(fig:life-cycle-label)). At 20$^\circ$C, 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 molting, 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 self-fertilised internally, 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) illustrates 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 can lay over a 1000 eggs a day which hatch within several hours. Dauer stage is a metabolically 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")
```

\newpage

### 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 behaviours.

#### Pharyngeal pumping

Pharyngeal pumping is the feeding behaviour 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. 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 allow for 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 to a series of electrical transients collectively called an EPG. These electrical transients are temporally defined and represent activities of distinct anatomical features of the phayngeal muscle, namely the corpus, isthmus and the teminal bulb [@raizen1994; @franks2006]. 

#### Locomotion ####{#locomotionbehaviour}

*C. elegans* exhibits distinct locomotory behaviour in liquid and on solid medium. Whilst in liquid it flexes back and forth in the middle of the body. On solid medium, it performs S shaped, crawling movement. The direction of this movement is mostly forward and achieved due to the friction between the substrate and the body [@niebur1993]. By counting the number of bends in the unit of time in the presence and absence of treatment, the effects on locomotory behaviour can be measured. 
 
#### Egg-laying ####{#egglayingbehaviour}

*C. elegans* reproduces mainly by self-fertilisation of hermaphrodites or less frequently by mating with males. Hermaphrodite is sexually ready to be fertilised from young adult. The eggs are stored in the uterus and laid in defined spacio-temporal fashion. Typically, 5 eggs are expelled from the vulva at the time in approximately 20 minute intervals [@waggoner1998]. The number of eggs laid in the unit of time can be counted and used to inform on the effects of treatment on the reproductive ability of the worm. 

### Nervous system and the neurotransmitters of *C. elegans*

One of the great advantages of *C. elegans* is that their entire nervous system has been mapped [@white1986] and the neurotransmitters underpinning its function identified. Electron microscopy of serial worm cross sections [@white1986] showed that a hermaphrodite has a total of 302 neurons present in the ventral nerve cord, the pharynx, the circumpharangeal ring and the tail. These neurons are assigned to 118 classes based on morphology and positioning. There are 39 sensory neurons, 27 motor neurons and 52 interneurons. Pharyngeal nervous system consists of 20 neurons belonging to 14 types.

The fast neurotransmission at the neuronal synapse and at the NMJ is mediated by a total of 6 neurotransmitters. Four of them are biogenic amines: octopamine, tyramine, dopamine and serotonin, which are involved in the modulation of many of the *C. elegans* behaviours.

Dopamine is synthesised in 8 mechanosensory neurons in hermaphrodites, whereas in males, it is present in those 8 neurons plus 6 tail mechanosensory neurons [@lints1999]. It is involved in the modulation of the locomotion. Specifically, it mediates a reduction in movement velocity upon initial food encounter [@sawin2000] and in the local exploratory behaviour in the vicinity of recently depleted food [@hills2004]. It is also involved in learning. It has been shown to regulate the onset of the accelerated forward movement in response to nonlocalised mechanical stimulus such as plate tapping [@sanyal2004], and adaptation to the odourants [@bettinger2004].

<!-- Four dopamine receptors have been identified to date: DOP1-DOP4, which are GPCRs [@sanyal2004; @chase2004; @chase2007]. -->

Tyramine and octopamine are minor neurotransmitters, which are present in a small number of cells. Octopamine is synthesised from tyramine in the gonad sheet cells and a pair of RIC interneurons [@alkema2005]. Its role is unclear, but it is potentially involved in the inhibition of egg-laying and pharyngeal pumping [@horvitz1982; @alkema2005]. Tyramine is synthesised in the same cells as octopamine as well as in a single pair of interneurons and gonad sheath cells, RIM motor neurons and the 4 uterine UV1 cells [@alkema2005]. It can act as an inhibitory neurotransmitter [@pirri2009] to prevent egg-laying and the head movement in response to anterior touch, as well as to modulate the reversal movement [@alkema2005]. 

5-HT (serotonin) functions in 8 types of neurons in *C. elegans* and has been shown to be involved in the regulation of locomotion, egg-laying and pharyngeal pumping. It is synthesised in a pair of Hermaphrodite Specific Neurons (HSNs) [@desai1988] and the VC4/5 neurons, which innervate vulval muscles [@duerr1999] and which upon release of 5-HT, stimulate egg-laying [@waggoner1998]. In the pharynx, it is released from the neurosecretory motor neurons (NSM) and the I5 interneurons [@chase2007], where it stimulates pumping in the presence of food [@horvitz1982; @avery1990; @sze2000] by acting on GPCRs SER1, SER4 and SER7 [@hobson2003; @song2013; @tsalik2003] and serotonin-gated chloride channel MOD-1 [@ranganathan2000]. More details on the role of 5-HT in the pharynx can be found in section \@ref(pharynx5ht). Serotonin is also involved in the modulation of locomotory behaviour, specifically enhanced slowing upon food encounter in food deprived animals [@sawin2000]. 

GABA acts primarily at the NMJ. It is present in a total of 26 neurons, which are motor neurons innervating the BWM, the head muscles and the enteric muscles, as well as interneurons [@schuske2004]. GABA serves both as excitatory and inhibitory neurotransmitter in *C. elegans*. It inhibits locomotion and foraging [@mcintire1993] (Section \@ref(locomotion)) by activating GABA~A~ ligand gated ion channels [@bamber1999] and induces enteric muscle contraction during defecation [@mcIntire1993] by acting on cation selective GABA-gated ion channel EXP-1 [@beg2003].

Glutamate is the most rapid excitatory and the second most frequently used neurotransmitter in *C. elegans* [@Serrano-Saiz2017]. It is present in 79 cells in hermaphrodites, and 98 cells males. Found in the head, pharynx, ventral cord, body and tail neurons [@loer2016]. In the pharynx, it is released from 4 neurons and it shortens the duration of the pump [@raizen1995]. More details on the function of glutamate in the pharynx can be found in section \@ref(glutamatepharynx). Glutamate also plays a role in the backing response upon nose touch [@mellem2002], the response to hyperosmotic stimuli [@Hilliard2002] and repellent odours [@hart1995]. It is also involved in the regulation of foraging behaviour [@hills2004] and long-term memory [@rose2003]. These functions are induced by acting on ionotropic glutamate gated ion channel (iGluR). There are at least 10 iGluR subunits in *C. elegans* [@brockie2003], some of which are excitatory, whereas others are inhibitory [@cully1994]. 

Like in the insect brain [@florey1963], ACh is a main excitatory neurotransmitter in the nervous system of *C. elegans*, where it is released from over a third of the *C. elegans* cells. In *C. elegans*, ACh is also a major neurotransmitter at the NMJ [@rand2007].

### Acetylcholine regulates feeding, locomotion and reproduction in *C. elegans* ### {#cholinergicneurotransmissioninworms}

Many of the *C. elegans* behaviours are regulated by acetylcholine, as evident from the behavioural analysis of mutant strains in which acetylcholine neurotransmission is affected. *C. elegans* cholinergic synapse expresses enzymes and transporters necessary for the cholinergic neurotransmission. Choline acetyltransferase (ChAT) encoded by the *cha-1* gene catalyses the formation of acetylcholine [@rand1985]. Vesicular acetylcholine transferase (VAChT) encoded by *unc-17* loads acetylcholine into synaptic vesicles [@alfonso1993]. Null mutations of these genes are lethal due to the inhibition of worm's locomotion and feeding and its eventual death due to starvation [@rand1989; @alfonso1993]. Polymorphic *ChAT* and *VAChT* mutants in which the expression is reduced, but not abolished, revealed somewhat opposite phenotypes. The pharyngeal pumping both in the presence and absence of food was reduced [@dalliere2015] the movement was highly uncoordinated and jerky [@rand1984], whereas egg-laying increased [@bany2003]. 

The role of cholinergic transmission was also investigated using pharmacological studies, in which the effects of cholinergic agents on the behaviour of worms were observed. Aldicarb is a synthetic carbamate commonly used as a nematicide [@lue1984] in the pest management systems. Its mode of action is via inhibition of the acetylcholine esterase (AChE) - the enzyme that breakdowns acetylcholine released to the synaptic cleft [@johnson1983]. When applied on worms, aldicarb causes hypercontraction of the BWM, leading to paralysis [@nguyen1995; @mulcahy2013], hypercontraction of the pharyngeal muscle and inhibition of feeding [@nguyen1995] as well as the inhibition of egg-laying [@nguyen1995]. These observations in conjunction with the phenotypical analysis of *cha-1* and *unc-17* mutants, suggest acetylcholine stimulates feeding, coordinates locomotion and inhibits egg-laying in *C. elegans*.

### *C. elegans* nAChRs ###{#celegansnacheintro}

Acetylcholine exerts its action by activating nAChRs. *C. elegans* contains 29 genes encoding for nAChR subunits [@jones2007a]. The receptor subunits are assigned to five groups based on the sequence homology: DEG-3, ACR-16, ACR-8, UNC-38, and UNC-26. The ECD domain sequence identity between members of these five groups and insect receptors is low (Figure (\@ref(fig:seqidentityecd-label)), suggesting distinct pharmacophores. 

(ref:seqidentityecd) **Amino acid sequence identity between the insect and *C. elegans* nAChR subunits.** Sequences of the honeybee and *C. elegans* extracellular, ligand binding domains were aligned using the Multiple Sequence Comparison by Log- Expectation (MUSCLE). Sequence identities were derived with the HMMER alignment and color-coded using red-yellow-green scale. *C. elegans* subunits of the UNC-38 group are the most homologous to the insect subunits.

```{r seqidentityecd-label, fig.cap = "(ref:seqidentityecd)", fig.scap="Amino acid sequence identity between the insect and \\textit{C. elegans} nAChR subunits", out.height = '120%', fig.align= 'center', echo=FALSE}
knitr::include_graphics("fig/general_intro/pdf/identity_clipped_renamed_aligned_celegans_apismelifera.png")
```

### Pharmacological evidence for the role of nAChRs in the regulation of *C. elegans* behaviour ###{#pharmacelegans}

Pharmacological experiments in which selective nAChR agonists, namely levamisole, nicotine and neonicotinoids, were tested against *C. elegans* behaviours provide evidence for the important role of these receptors in the regulation of locomotion, pharyngeal pumping and egg-laying. 

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

Nicotine is an alkaloid naturally occurring in the *Tobacco* plant [@steppuhn2004]. It is an agonist of the second type receptor at a BWM, namely the N-type [@ballivet1996], but based on the nicotine-intoxication worm phenotype, it is also likely to target receptors regulating pharyngeal pumping and vulva muscle. Nicotine inhibits locomotion and egg-laying [@kudelska2017]. It also inhibits pharyngeal pumping in the presence of food [@kudelska2018], whereas in the absence of food, it stimulates pharyngeal pumping [@raizen1995]. 
<!-- There is a limited literature regarding the effects of neonicotinoids on nematodes. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of neonicotinoids. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014]  -->

<!-- Thiaclopand inhibits its egg hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. -->
nAChR agonists neonicotinoids have been shown to affect motility [@hopewell2017; @mugova2018] and egg-laying [@gomez-amaro2015; @ruan2009] (Section \@ref(chapter3effectsofneonics)).

### Genetic evidence for the role of nAChRs in the regulation of *C. elegans* behaviour ###{#pharmacelegans}

Behavioural analysis of *C. elegans* mutants supports the role of nAChRs in egg-laying, locomotion and pharyngeal pumping and reveals further functions of these receptors. Several mutant strains, including *unc-29*, *unc-38* and *unc-63* [@lewis1980b] as well as *unc-29; acr16* and *unc-63; acr-16* double mutants [@touroutine2005] exhibit disrupted, highly uncoordinated movement. *unc-29*, *unc-38* and *unc-63*, *lev-1*, *lev-8* mutants exhibits markedly inhibited egg-laying in the presence of levamisole [@waggoner2000]. *C. elegans eat-2* mutant shows 70% reduction in the pumping rate in the presence of food [@raizen1995; @mckay2004]. @yassin2001 has shown that *deg-3* mutants are deficient in choline-chemotaxis, suggesting this subunit plays a role in sensory transduction. In the *unc-63* mutant, the action of nAChR agonist DMPP, which delays cell divisions and differentiation during the L2 developmental stage in hindered [@ruaud2006] suggesting a role of this subunit in the development of *C. elegans*.

### Stoichiometry of *C. elegans* nAChRs ####{#muscletypenachr} 

*C. elegans* expresses at least 29 nAChR subunits (Section \@ref(celegansnacheintro)), which are expressed at the neuromuscular junction (NMJ) [@richmond1999] and in the nervous system [@lewis1987]. To date, four receptor assemblies have been identified. (1) A single neuronal receptor composed of DES-2 and DEG-3 subunits [@treinin1998]. (2) There are two receptor at the BWM differentiated based on their pharmacology into L-(levamisole) type and N-(nicotine) type [@richmond1999]. EAT-2 is a predicted $\beta$ nAChR subunit expressed in the pharyngeal muscle, believed to assemble with auxilary subunit EAT-18 [@mckay2004]. 

<!-- To date, four receptor assemblies have been identified [@richmond1999; @treinin1998; @touroutine2005]. Two of the *C. elegans* nAChRs are expressed at the post-synaptic membrane of the neuromuscular junction of the BWM [@richmond1999]. The identity and function of these proteins has been studies using a combination of behavioural, pharmacological and electrophysiological approaches. -->
\newpage

##### L-type receptors

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

##### N-type receptors 

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

#### Receptors at the pharyngeal neuromuscular junction ####{#eat2atthepharynx}

A single receptor subunit, namely EAT-2 has been identified as essential in mediation of the feeding response [@mckay2004]. *C. elegans eat-2* mutant shows significantly reduced pumping in the presence of food [@raizen1995; @mckay2004]. A similar phenotype was noted in the *eat-18* mutants. EAT-18 however is not a nAChR subunit. Instead, it is predicted to be a single transmembrane protein. Based on the localisation and behavioural phenotype, EAT-18 and EAT-2 are believed to co-assembly to form a functional receptor [@mckay2004]. 

ACR-7 is also expressed at the pharyngeal muscle [@saur2013], as was shown with a reporter construct, however its function in pharyngeal pumping is unclear as *acr-7* mutant pump normally in the presence of food [@saur2013].

### *C. elegans* proteins important in nAChR maturation ###{#cematnachr}

The function of nAChRs in *C. elegans* depends on the successful receptor maturation, a process aided by a number of chaperon proteins. 
<!-- Identification and heterologous expression of *C. elegans* nAChRs greatly contributed to the understanding of proteins involved in the biogenesis of these ion channels. There are two nAChRs expressed at the NMJ of the body wall muscle of *C. elegans*: ACR-16 homopentamic receptor and a heteropentameric receptor composed of UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8. These receptors are classified based on pharmacology into nicotine- and levamisole- sensitive and named N-type and L-type, respectively. DEG-3/DES-2 nAChRs are the neuronal receptors [@treinin1998], whereas EAT-2 containing nAChRs are expressed in the pharyngeal muscle [@mckay2004].  -->

<!-- *C. elegans* is a powerfull tool in which the function and pharmacological properties of proteins can be studied by a combination of genetic, behavioural and cellular approaches (Section \@ref(genmanip) \@ref(analytical_behaviour)). -->

#### RIC-3 ####{#ric-3celegans}

RIC-3 is a evolutionary conserved, ER-residing [@roncarati2006; @alexander2010] TM protein [@wang2009]. In *C. elegans*, it is ubiquitously expressed in most (if not all) neurons, and in the pharyngeal and BWMs [@halevi2002]. The predicted topology of *C. elegans* RIC-3 has 2 transmembrane domains and 3 coiled-coils. The *C. elegans ric-3* mutant has impaired locomotor behaviour, resistance to levamisole [@miller1996] and impaired responses to cholinergic agents, as measured by electrophysiological recording from the BWM [@halevi2002]. The *C. elegans ric-3* mutant has impaired cholinergic neurotransmission; there is a lack of cholinergic component of the EPG recording resulting in significantly retarded pharyngeal pumping and starved appearance [@halevi2002].

Heterologous expression of *C. elegans* nAChR in *Xenopus* oocytes provides evidence for their function in receptor maturation. Choline-evoked currents of neuronal DEG-3/DES-2 and muscle ACR-16 receptors increased upon RIC-3 co-expression [@halevi2002; @ballivet1996]. The role of RIC-3 was also demonstrated *in-vivo*. Nicotine induced current at the BMW was markedly reduced in *ric-3* mutant, in comparison to wild-type [@halevi2002]. 

*C. elegans* RIC-3 can also promote maturation of mammalian $\alpha7$ channels. Co-expression of this protein improved $\alpha7$ function in *Xenopus* oocytes as shown by enhanced choline- and acetylcholine- evoked currents and cell-surface binding of radiolabelled $\alpha$-bgtx [@lansdell2005; @williams2005]. RIC-3 also enabled expression of $\alpha7$ in otherwise non-permissive insect cell lines [@lansdell2008]. It has been shown to not only promote the heterologous cell-surface expression of mammalian receptors, but it also increased the expression of insect chimera nAChRs [@lansdell2012].

#### UNC-50 ####{#unc50}

UNC-50 is an ortholog of evolutionary conserved GMH1 protein. In *C. elegans* it was first identified in behavioural and pharmacological screens of *C. elegans* mutants. Several phenotypes have been described including: uncoordinated movement [@lewis1980] reduced binding of radiolabelled levamisole to the membrane fractions [@lewis1987], resistance to levamisole in behavioural assays [@lewis1987; @abiusi2017] and no responses of L-type nAChRs at the BWM to levamisole [@eimer2007]. The lack of cell-surface staining from antibodies against UNC-29 [@eimer2007] in *unc-50* mutant confirmed the role of UNC-50 in nAChR maturation. *unc-50* mutant is also characterised by an increased lysozyme-dependent degradation of nAChRs, suggesting its preventative role in this process. UNC-50 is predicted to be expressed in the Golgi, as the expression of GFP::UNC-50 fusion protein resulted in fluorescence typical of the localisation to this organelle [@eimer2007].

#### UNC-74 ####{#unc74}

UNC-74 is closely related to the human TMX3 protein which is thought to be ER-associated [@haugstetter2005]. Reduced radiolabelled meta-aminolevamisole binding to membrane fraction of *C. elegans* mutant [@lewis1987] combined with its role in expression of L-type receptor in *Xenopus* oocytes [@boulin2008] confirms its role in receptor maturation. 

#### EAT-18 ####{#eat18}

EAT-18 is thought to be required for the function of pharyngeal nAChRs. It consists of a single transmembrane and an extracellular domain. Transgenic worms expressing EAT-18::GFP fusion protein reveal fluorescence in the pharynx with the strongest signal in the muscle, but also in the pharyngeal neuron M5 and unidentified 5 to 6 extrapharyngeal neurons [@mckay2004]. *eat-18* mutants are deficient in pumping and resistant to high concentration of nicotine, supporting the function of EAT-18 in cholinergic neurotransmission of the pharynx [@raizen1995]. The association of *eat-18* with pharyngeal nAChR was indicated by comparison of the staining in the wild-type and *eat-18* mutant strains. Injection into the pseudocoelom of radiolabelled $\alpha$-bgtx resulted in straining of the pharynx. This was however abolished in the mutant strain [@mckay2004]. In addition, the expression of EAT-2 in *eat-2* mutant was normal, suggesting EAT-18 is not involved in the trafficking of this receptor. It has been proposed that EAT-18 co-assembles with EAT-2 due to their common pharyngeal phenotypes in mutant strains and common cellular localisation in the pharyngeal muscle [@mckay2004]. Recently, successful expression of eat-2 co-assembled with eat-18 has been shown in *Xenopus* oocytes (personal communication).

<!-- # ```{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) -->
<!-- # ``` -->

### Mode of action studies in *C. elegans* 

The depth of *C. elegans* genetic, anatomical, biochemical and pharmacological knowledge combined with methods to generate mutant and transgenic lines, provides an opportunity to use this organism in the mode of action studies of cholinergic agents. Indeed, *C. elegans* has been utilised to investigate the molecular basis of biocides selectivity, including nAChR levamisole [@pinnock1988; @lewis1987; @lewis1980]. *C. elegans* is also a promising model in which functional and pharmacological properties of nAChRs can be studied. 
 
<!-- #### Mode of action studies in mutant *C. elegans* -->

<!-- There is a range of genetic techiques available to generate mutant strains, in which the expression of a certain protein is greatly reduced or eliminated [@boulin2012]. Using these techniques, hundreds of mutant strains have been peoduced. These strains have been deposited and are available from the *Caenorhabditis* Genetics Center (CGC). Over the years, behaviour analysis of wild-type and mutant strains in the -->


#### Pharmaceutical characterisation of proteins in transgenic worms ####{#pharmaintrangenicworms}

A process of microinjection allows for generation of transgenic worms in which heterologous expression can be achieved. Microinjection is a process by which a plasmid containing cDNA encoding for a protein of interest is injected into the syncytium distal arm of the gonad(s) of the young adult hermaphrodite worm [@stinchcomb1985]. The injected DNA in taken up by the the residing oocytes [@wolke2007], which become fertilised and develop into adult worms. Using cellular machinery, the DNA plasmid forms extrachromosomal arrays, from which the cDNA becomes transcribed, translated and expressed [@stinchcomb1985; @mello1991]. 

The expression of transgene can be driven in specific cells or tissues, including those reliant on cholinergic transmission. Conjugated monoclonal antibodies were used to show selective expression of myo-3 (heavy chain of myosin B) at the body-wall muscle and vulva muscle [@ardizzi1987] and myo-2 (myosin heavy chain C) in the pharyngeal muscle [@okkema1993] of the intact worm. Thus, by using myo-3 or myo-2 promoters upstream of the heterologous gene, expression at the body wall or pharyngeal muscle, respectively, can be achieved [@sloan2015; @crisford2011]. There are also promoters, such as H2O, inducing expression in the nervous system [@yabe2005]. Behavioural and cellular analysis of transgenic worms generated by microinjection, allows for pharmacological characterisation of native and exhogenous proteins, including nAChRs [@sloan2015].

### Biochemical methods to assess expression of nAChR in *C. elegans* ###{#biochemapproachesforexpression}

The cellular localisation of nAChRs expressed in *C. elegans* can be detected by an array of methods, such as using protein-specific pharmacological agents. $\alpha$-bgtx is a high affinity antagonist of nAChRs [@blumenthal1997], widely used to label expression on native and heterologous channels. Audioradiography of tissues incubated with radiolabelled $\alpha$-bgtx visualised mammalian nAChRs at the post-synaptic membrane of the end-plate [@barnard1971], in the peripheral [@clarke1985] and central nervous system [@carbonetto1979]. Fluorescently labelled $\alpha$-bgtx was utilised to show successful expression of heterologous proteins such as mammalian $\alpha7$ in HEK, P12 and SH SY5Y cell lines [@cooper1997; @gu2016]. In *C. elegans*, conjugated-$\alpha$-bgtx injected into the pseudocoelom, labelled native nAChRs of the pharyngeal [@mckay2004] and BWM [@jensen2012]. It also allowed for the identification of heterologously expressed ACR-16 in the BWM of *C. elegans* [@jensen2012]. $\alpha$-Bgtx is used to demonstrate cell surface expression, because it binds to the extracellular domain of the nAChR [@dellisanti2007] and does not permeate membranes. There are methods used to label heterologous proteins intracellularly. For example, @salom2012 and @gu2016 used detergents to permeabilised membrane to allow protein-specific antibodies or $\alpha$-bgtx to access protein sites inside the cell.

Taken together, *C. elegans* is an attractive biological system in which the mode of action and selective toxicity of cholinergic agents can be studied. It is also a promising biological system for the heterologous expression of nAChRs, in particularly those that do not express well in other biological systems. Generally speaking, heterologous expression of proteins can have two consequences: (1) when introduced into the mutant strain, it can restore drug or cellular function [@crisford2011; @salom2012] and (2) heterologous expression in wild-type worm can lead to new or altered pharmacological sensitivity [@crisford2011; @salom2012]. These changes can be scored using an array of behavioural and cellular methods. Therefore, heterologous expression combined with behavioural and pharmacological analysis of transgenic worms can also inform on functional and pharmacological properties of heterologously expressed proteins.

<!-- For example, expression of *C. elegans* and human ortholog of the potassium-activated calcium channel Slo-1 at the body wall muscle of *C. elegans slo-1* mutant restored sensitivity to selective agonist emodepsite in locomotory assays  -->

<!-- emonstrated that the ectopical expression of Slo-1 in the pharyngeal muscle cell confers sensitivity to emodepsite. -->

<!-- The locomotory deficit and levamisole-resistance of *C. elegans unc-38* mutants was reversed upon expression of nAChRs of the parasitic worm UNC-38 [@sloan2015]. Whereas human GPCR A~2a~R expressed in the body wall muscle or nerve cells conferred behavioural response to receptor specific ligand [@salom2012]. In addition, the A~2A~R selective agonist CGS21680 stimulated locomotion of transgenic worms in a dose dependent manner. Enhanced response to non-specific GPCR agonist adenosine in transgenic worms expressing A~2a~R in the body wall muscle was also noted.  -->

<!-- Heterologous expression of nAChRs can have several consequences, due to their involvement in many biological processes. *C. elegans* nAChRs are the main excitatory receptors at the neuromuscular junction [@riddle1997) and are involved in regulation of many aspects of worm’s behaviour, including feeding [@avery2012] locomotion [@richmond1999], egg-laying [@schafer2005; @bany2003]. They are also expressed in neuronal cells constituting to sensory and integrating circuits and are involved in chemosensory behaviour, as well as signal integration and neuronal plasticity [@yassin2001]. More recently their implicated in the regulation of development has been investigated [@ruaud2006].  -->

### Aims

The overall aim of this project is to develop *C. elegans* as a platform for the heterologous expression of nAChRs, with the aim to gain insight into selective toxicity of neonicotinoids insecticides. This will be achieved in three steps:

1. Define sensitivity of *C. elegans* to these compounds. The representatives of three distinct chemical classes of neonicotinoids will be used: cyanoamidine clothianidin, nitroguanidine thiacloprid and nitromethylene nitenpyram. Their effects on *C. elegans* will by tested utilising behavioural and cellular assays to define their potency on distinct neuronal circuits. 

2. Identify suitable *C. elegans* genetic background with defined cholinergic function for the expression of nAChRs.

3. Develop assays by which the functional nAChR expression and drug-sensitivity can be tested.