Commit f973b6ce authored by mk11g11's avatar mk11g11
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parent faea1e38
\phantomsection
\pdfbookmark[0]{Title Page}{title}
<!-- Title, name and date -->
\title{\LARGE {\bf My awesome thesis dissertaion title}\\
\title{\LARGE {\bf Investigation of the selective toxicity of neonicotinoids using the nematode worm Caenorhabditis elegans}\\
\vspace*{6mm}}
\author{Monika Kudelska}
\maketitle
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......@@ -7,28 +7,29 @@ nocite: |
## Chemical treatment in agriculture
Insecticides are compounds utilised in agriculture, medicine, industry and private households to protect crops, life-stock and human health from pest infestation. Their identity evaluated over the years to improve the effectiveness and reduce the undesirable effects on human health and the environment.
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]. Their identity evaluated over the years to improve the effectiveness and reduce the undesirable effects on human health and the environment [@casida1998].
Until late 1800s organic, natural compounds contained within the plant or animal matter were utilised [@agarwal2006]. For example, tobacco plant containing nicotine was applied in France to protect pear orchards against bug lice, whereas *Chrysanthemum* plants containing pyrethrum were used against worms and insects in America and Europe. These treatment were however suitable only for small scale agricultural treatment.
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, has been used in France, England and the US 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 synthetic 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].
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 use 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. DTT 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]. DTT 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 pyrothroids (Table \@ref(tab:insecticidegroups)). By the 1990s, the respective market share of members of these three classes of insecticides was: 43 %, 15 % and 16 % [@jeschke2011]. The main issue associated with the use of organophosphates and carbamates is their ability to cause serious human poisonings, some of 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.
Diminishing popularity of DDT, created a market space for organophosphates, carbamates and pyrothroids (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 bn Euros, 1.19 bn Euro and 1.169 bn Euro, respectively [@jeschke2011]. The main issue associated with the use of organophosphates and carbamates is their ability to cause serious human poisonings, some of 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"),
Marketed = c("1930s-current", "1951-current", "1949s-current"),
Sales = c("3.42 bn", "1.19 bn", "1.169 bn"),
Chemicals = c("Parathion, malathion, azinphosmethyl", "Aldicarb, carbamyl, carbofuran", "Allethrin, Cypermethrin"))
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,
caption = 'Synthesis insecticides insectides') %>%
col.names = linebreak(c("Class", "Chemical", "Mode of\naction")),
caption = 'Synthetic insecticides') %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote("Sales as of 2008, according to Jeschke et al. 2011",
threeparttable = T)
......@@ -38,30 +39,34 @@ library(kableExtra)
### Synthesis
In 1970s, the scientists of Shell Development Company Biological Research Centre in California identified alpha- DBPN (2-(dibromonitromethyl)-3-(methylpyridine)), first synthesised by Prof. Henry Feuer [@feuer1986]. This lead compound showed low insecticidal activity against aphid and house fly [@tomizawa2003; @tomizawa2005]. Structural alterations of DBPN resulted in production of nithiazine (Figure \@ref(fig:neonics-structure-label)). Nithiazine showed improved insecticidal activity and was particularly effective as a new housefly repellent [@kollmeyer1999]. Further replacement of the thiazine ring by chloropyridinylmethyl (CPM) group, addition of the imidazolidine or its acyclic counterpart, and retention of the nitromethylene group resulted in generation of more potent compounds, one of which, nitenpyram, exhibited particularly high efficacy. Regrettably, both nithiazine and nitenpyram are not useful in fields, as they substantially absorb in the sunlight, resulting in their degradation and loss of activity against pests. The latter however is successfully used in veterinary medicine as an external parasite treatment for cats and dogs.
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 was replaced by nitroguanidine and cyanoamidine [@kagabu1995]. These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
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 [@pan2019] whereas their mis-classification has been deliberate to avoid association with neonicotinoids.
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 synthetic insecticides neonicotinoids.** Systematic modification of the lead and prototype compounds led to the discovery of seven neonicotinoids currently used in agriculture and animal health. They are classified according to the pharmacophore moiety into N-nitroguanidines, N-cyanoamidines and nitromethylenes.
(ref:neonics-structure) **Development and chemical structures of synthetic insecticides neonicotinoids.** Systematic modification of the lead and prototype compounds led to the discovery of seven neonicotinoids currently used in agriculture and animal health. They are 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 = '60%', echo = FALSE}
```{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 ###{#economicalstatus}
The use of neonicotinoids in agriculture has been increasing steadily since the market launch in the early 1990s. By 2008, they became major players in the agriculture [@jeschke2011], 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 raise in their economical impact. In 2008, the estimated global market value of neonicotinoids was 1.5 bn dollars [@jeschke2011]. This increased to 3.1 bn dollars in 2012 [@bass2015].
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 bn dollars [@jeschke2011]. This increased to 3.1 bn dollars in 2012 [@bass2015].
The raising usage and monetary value of neonicotinoids is a reflection of their many advantages.
The widespread usage and monetary value of neonicotinoids is a reflection of their many advantages.
<!-- Important in the pest managment, used in over 120 coutries on 140 crop types [@jeschke2011]. -->
## Physicochemical properties ##{#physchem}
### Properties ##{#physchem}
One of the major benefits of neonicotinoids are their beneficial physical and chemical property profiles (Table \@ref(tab:properties)), therefore are stable in the environment and can be applied by a diversity of methods. Due to relatively high water solubility, they act as systemic insecticides [@westwood1998]. This means that once applied on crops, neonicotinoids dissolve in the available water and can be taken up by the developing roots or leaves. Upon plant entry, they are then distributed to all parts of the plant [@haas2006], providing protection against herbivorous pests. This systemic property of neonicotinoids means they can be used as a seed coating, reducing the required frequency of application. Indeed, seed dressing is the most commonly used method, accounting for 60 % of all neonicotinoids applications worldwide [@jeschke2011] and particularly popular to protect potatoes, oilseed rape, cereal, sunflower and sugar beet. In addition, neonicotinoids half-life in soil is from several weeks to years [@cox1997; @sarkar2001; @gupta2007), hence seed-dressing creates a continual source for re-uptake by plants. They are also suitable for ground treatment and are used as soil drenching for the protection of citrus trees and vines, granules for amenity grassland and ornament flowers and as a trunk-injection to protect trees against herbivores. Low vapor pressure and low Henry’s Law constant of neonicotinoids means they are not volatile, therefore can be applied as spray. This method is used in garden for flowers and vegetables and in agriculture on soft fruits and greenhouse crops. Low lipophilicity, indicated by octanol/water partition coefficient value (log Pow), suggest they do not bio-accumulate in the adipose tissues [@turaga2016]. However, moderate water solubility combined with low lipophilicity means they may have a potential to accumulate in water.
One of the major benefits of neonicotinoids are their physical and chemical profiles (Table \@ref(tab:properties)).
Although structurally related nicotine has similar properties, it is not appropriate for the agricultural use due to low toxicity to insects [@nauen1996].
#### Diverse methods of applications
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 can be 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 garden for flowers and vegetables and in agriculture on soft fruits and greenhouse crops. Low lipophilicity, indicated by octanol/water partition coefficient value (log Pow), suggest they do not bio-accumulate in the adipose tissues of animals [@turaga2016]. However, moderate water solubility combined with low lipophilicity means they may have a potential to accumulate in water.
<!-- Although structurally related nicotine has similar properties, it is not appropriate for the agricultural use due to low toxicity to insects [@nauen1996]. -->
```{r properties, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
......@@ -84,11 +89,11 @@ library(kableExtra)
threeparttable = T)
```
### Highly potent against insect pests ###{#potentpests}
#### 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, as measured in the acute toxicity assays. During those assays, animals were dosed orally, systemically or topically with various concentrations of drugs. The number of dead animals was scored to derive the LC50/LD50 (the concentration/dose of a compound that kills 50 % of the population). The lower the LC(D)50, the greater the potency of a compound.
Neonicotinoids are highly potent against insect pests, as measured by the LC50/LD50 (the concentration/dose of a compound that kills 50 % of the population) in the acute toxicity assays (Table \@ref(tab:toxallanimal)). The lower the LC(D)50, the greater the potency of a compound.
Neonicotinoids are effective against a wide range of piercing-sucking pests such as cotton and peach aphids (**Aphis gossypii** and *Myzus persicae*) [@nauen1996; @mota-sanchez2006; @bass2011], domestic (*Malus domestica*) and may-flies (*Epeorus longimanus*) [@tomizawa2000; @alexander2007] as well as planthoppers (*Nilaparvata lugens*) [@zewen2003]. Their IC50 is in generally 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* was clothianidin > nitenpyram = thiacloprid, suggesting nitroguanidines are generally more potent than nitromethylenes and cyanoamidines [@shi2011; @mota-sanchez2006].
......@@ -97,7 +102,7 @@ The potency also depends on the route of exposure. LC50s are lower upon systemic
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 tox-all-animal-labels, echo=FALSE, warning = FALSE, message = FALSE}
```{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"
......@@ -140,11 +145,11 @@ library(dplyr)
# to calculate the ng/mg cocroach divided by 175, bee by 100
```
### Selectively toxic to insect pests. ###{#seltox}
#### Selectively toxic to insect pests. ###{#seltox}
One of the key determinants of success of agrochemical compounds is their ability to selectively target insects over non-target species. Neonicotinoids are generally effective at ~ 2 $\mu$M concentrations against piercing-sucking pest infestations, whereas their LD50s is 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 is at least 2 times higher. Honeybees (*Apis mellifera*) are among the most susceptible non-targets, with the average LC50 and LD50 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 LC50 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 LD50 doses higher than 130 mg/kg of body weight [@decant2010; @legocki2008]. This differential susceptibility between target and non-target species, in 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 (i.e. worms and honey bee).
One of the key determinants of success of agrochemical compounds is their ability to selectively target insects over non-target species. Neonicotinoids are generally effective at ~ 2 $\mu$M concentrations against piercing-sucking pest infestations, whereas their LD50s is 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 is at least 2 times higher (Table \@ref(tab:toxallanimal)). Honeybees (*Apis mellifera*) are among the most susceptible non-targets, with the average LC50 and LD50 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 LC50 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 LD50 doses higher than 130 mg/kg of body weight [@decant2010; @legocki2008]. This differential susceptibility between target and non-target species, in 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 (i.e. worms and honey bee).
Concentrations of neonicotinoids in nectar, wax and pollen was been investigated. Generally, the highest concentrations are found in the former [@goulson2013]. @cresswell2011 determined that the 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 LC50 and LD50 values of 7.04 $\mu$M and 4.5 ng, respectively [@@cresswell2011].
Concentrations of neonicotinoids in nectar, wax and pollen was been investigated. Generally, the highest concentrations are found in the former [@goulson2013]. @cresswell2011 determined that the 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 LC50 and LD50 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 LC50 against the most susceptible species is 2.74 $\mu$M [@luo1999].
......@@ -161,38 +166,36 @@ The concentration of neonicotinoids in soils with several years of history of tr
### Sub-lethal effects of neonicotinoids on non-target species ###{#sublethal}
#### Insect pollinators
#### Insect pollinators ####{#sublethalbees}
Although field realistic neonicotinoids are not expected to kill bees, a substantial body of evidence from lab- and field- based experiments suggest that they can impair on the cognitive function and reproduction of these biological pollinators.
Pollinating services are provided by many species of bees, flies, beetles and bats [@thapa2006]. Eightly percent of the total pollinating activity is carried out by bees [@thapa2006]. There are over 20 000 species of bees, 267 of each life in the UK [@breeze2012a]. Among them are honeybees (*Apis mellifera*, *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. Although field realistic neonicotinoids are not expected to kill bees, a substantial body of evidence from lab- and field- based experiments suggest that they can impair on the cognitive function and reproduction of these biological pollinators.
##### Reduced olfactory learning and memory
Bees are social insects, living in colonies where a clear division of labor exists. Worker bees account for ~ 90 % of the entire colony. 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 and memorise sensory cues is crucial for the survival and overall success of the entire hive.
The ability of bees to olfactory learn can be measured with Proboscis Extension Reflex (PER) paradigm. PER conditioning is a process by which an insect learns to extend its tongue (proboscis) in response to olfactory stimuli, typically evoked by the sugar solution contacting the antenna [@takeda1961]. Alternatively, their olfactory learning capability can be measured by scoring various aspects of foraging behaviour. Laboratory and semi-field studies show that many aspects of worker bees' behaviour is impaired by neonicotinoids.
Bees exposed to 93 nM of imidacloprid in the sugar solution showed reduced ability to olfactory learn, as showed by the PER [@decourtye2004]. Imidacloprid also compromised foraging activity of honeybees [@decourtye2004; @gill2012] and bumblebees. 4- week exposure of early-developmental stages to imidacloprid at 23 nM in pollen reduced the foraging efficiency and duration [@gill2012]. Neonicotinoids also reduce the number of bees returning to hives. Increased home failure were noted in honeybees exposed to a single dose of thiamethoxam at 1.35 ng [@henry2012] and in bumblebees fed diet containing imidacloprid at 23 nM and 3 nM for 2 weeks [@feltham2014].
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. The olfactory learning can be measured with Proboscis Extension Reflex (PER) paradigm. PER conditioning is a process by which an insect learns to extend its tongue (proboscis) in response to olfactory stimuli, typically evoked by the sugar solution contacting the antenna [@takeda1961]. Alternatively, their olfactory learning capability can be measured by scoring various aspects of foraging behaviour. Bees exposed to 93 nM of imidacloprid in the sugar solution showed reduced ability to olfactory learn, as showed by the PER [@decourtye2004]. Imidacloprid also compromised foraging activity of honeybees [@decourtye2004; @gill2012] and bumblebees. 4- week exposure of early-developmental stages to imidacloprid at 23 nM in pollen reduced the foraging efficiency and duration [@gill2012]. Neonicotinoids also reduced the number of bees returning to hives. Increased home failure were noted in honeybees exposed to a single dose of thiamethoxam at 1.35 ng [@henry2012] and in bumblebees fed diet containing imidacloprid at 23 nM and 3 nM for 2 weeks [@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]. --> -->
<!-- <!-- 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
The 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.
The 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. Neonicotinoids have been shown to negatively impact on various aspects of bees' fecundity. 14-days exposure of bumblebees to imidacloprid at 2, 4 and 23 nM, increased 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]. 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].
Neonicotinoids negatively impact on various aspects of bees' fecundity. 14-days exposure of bumblebees to imidacloprid at 2, 4 and 23 nM, increased 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].
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].
Negative impact of neonicotinoids is 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].
#### soil worms ####{#sublethalsoilworm}
Soil worms includes segmented worms representing Phylum Annelida and the round worms from Nematoda phylum. The segmented worms include earth dwellers such as redworm *Eisenia fetida* and the common earthworm *Lumbricus terrestris*. All annelids are free-living. Nematodes on the other hand are classified as parasitic and non-parasitic. *C. elegans* is an example of a non-parasitic worm, which feeds on bacteria associated with rotting fruits or vegetables. Soil parasitic worms can be divided into those that infect plans or animals. They can cause large health, life-stock and agricultural loses. For example, *Melyidogyne* species including *Meloidogyne incognita*, account for 95 % of all plant infestation species and cause a loss of 5 % of global crops [@taylor1978] including loss tomatoes, cowpea and blackpepper.
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].
To investigate the effects of neonicotinoids on soil dwellers, worms were exposed to various concentrations of drugs in solution, or in artificial soils. The concentrations effective against various behaviours and parameters such as fecundity, body weight, locomotion, sensory processing and burrowing were noted.
#### soil worms
##### Earth worms
Clothianidin and thiacloprid at concentrations $\ge$ than 1.2 $\mu$M and the EC50 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* [@alves2013]. Reduction of body weight of *E. fetida* and *D. octaedra* were observed after 14-day treatment with imidacloprid at 27.08 and 54.75 $\mu$M [@kreutzweiser2008]. Imidacloprid at 488.85 nM to 7.82 $\mu$M increased avoidance of *E. andrei* [@alves2013], 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].
To investigate the effects of neonicotinoids on soil dwellers, worms were exposed to various concentrations of drugs in solution, or in artificial soils. The concentrations effective against various behaviours and parameters such as fecundity, body weight, locomotion, sensory processing and burrowing were noted.
##### Soil nematodes
Clothianidin and thiacloprid at concentrations $\ge$ than 1.2 $\mu$M and the EC50 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* [@alves2013]. Reduction of body weight of *E. fetida* and *D. octaedra* were observed after 14-day treatment with imidacloprid at 27.08 and 54.75 $\mu$M [@kreutzweiser2008]. Imidacloprid at 488.85 nM to 7.82 $\mu$M increased avoidance of *E. andrei* [@alves2013], 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]. Neonicotinoids also induce a sublethal effect on the the free-living nematode *C. elegans*. Thiacloprid and imidacloprid have an effect on the reproduction of *C. elegans* with EC50 of 1.14 nM and 2.09 mM, respectively [@gomez-eyles2009]. Thiacloprid at 37 nM has an effect on chemosensing, whereas at 18 $\mu$M it impairs motility of this free living nematode [@hopewell2017]. Impaired motility of *C. elegans* in response to $\ge$ 120 $\mu$M imidacloprid was also recorded [@mugova2018]. Taken together, neonicotinoids have sublethal effects on earth worms and soil nematodes at concentrations as low as nM.
Neonicotinoids also induce a sublethal effect on the the free-living nematode *C. elegans*. Thiacloprid and imidacloprid have an effect on the reproduction of *C. elegans* with EC50 of 1.14 nM and 2.09 mM, respectively [@gomez-eyles2009]. Thiacloprid at 37 nM has an effect on chemosensing, whereas at 18 $\mu$M it impairs motility of this free living nematode [@hopewell2017]. Impaired motility of *C. elegans* in response to $\ge$ 120 $\mu$M imidacloprid was also recorded [@mugova2018]. Taken together, neonicotinoids have sublethal effects on earth worms and soil nematodes at concentrations as low as nM.
Most of the doses effective against worms are higher than the average doses of neonicotinoids in the field. However, the presence of clothianidin, imidacloprid and thiamethoxam has been detected at lower than average levels, such as 80.10 nM for imidacloprid, 23.01 nM for imidacloprid and 68.56 nM for thiamethoxam [@jones2014]. This suggests that the environmentally relevant concentrations of neonicotinoids may negatively impact on the the well-being of soil dwellers.
......@@ -200,7 +203,7 @@ Most of the doses effective against worms are higher than the average doses of n
#### Birds
Evidence suggests that environmental relevant concentrations of neonicotinoids may have negative effects on birds [@hallmann2014]. In particular, granivorous and insectivorous vertebrates may be at risk, should they consume neonicotinoid-contaminated seeds and/or insects [@goulson2013]. Environmental neonicotinoids may impair migratory ability [@eng2017] and negatively impact on the growth and reproduction [@sanchez-bayo2016] of birds.
Evidence suggests that environmental relevant concentrations of neonicotinoids may have negative effects 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.
......@@ -216,13 +219,13 @@ The environmental ecotoxicity of neonicotinoids highlights the importance of sel
<!-- knitr::include_graphics("fig/general_intro/png/nAChR_topology_3.png") -->
<!-- ``` -->
### nAChR structure ###{structure}
### nAChR structure ###{#structure}
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 type A (GABA) 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(structure-nachr-label)a) shed light on the the stechiometry, the shape and the size of Cys-loop 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 type A (GABA) 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 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. 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-PAGE pattern and the analysis of nAChR complexes purified with the use of non-denaturing buffer led to a suggestion that the stechiometry 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 were either absent or greatly reduced, therefore 4 subunits are required for the normal function of this protein [@mishina1984].
The stechiometry 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. Constituting nAChR subunits are arranged pseudosymmetrically, around the central ion conduction pore [@brisson1985]. The subunit composition of the neuromuscular nAChR follows the strict order of $\alpha1$, $\beta1$, $\delta$, $\alpha1$, $\gamma$ (clockwise). Each subunit of the nAChR contains 4 transmembrane helices [@noda1982; @noda1983] named M1, M2, M3 and M4, as moving from N- to C- terminus. M1, M3 and M4 are exposed to the plasma membrane [@blanton1994], shielding M2, pore-forming helices [@imoto1986; @hucho1986] from the hydrophobic environment of the bilayer. As the outer helices progress from the outer to the inner leaflet of the membrane, they tilt inwards [@miyazawa2003], narrowing down the width of the channel. M2 on the other hand, bends roughly in the middle of the bilayer [@unwin1995], where it forms the most restricted part of the ion conductivity pathway. There are hydrophobic interactions between the outer helices, which stabilise the outer wall of the receptor and hence limit the conformational changes adopted by the inner helix. In contrast there are no extensive bonds between the inner and outer helices [@miyazawa2003]. As lining pore structures, the inner helix and flanking sequences contain molecular determinants for ion selectivity, permeability, the rate of conductance and gating. These were investigated by pharmacological, biochemical and electrophysiological approaches. [@imoto1988; @imoto1991; @konno1991] investigated the function of several rings of anionic and neutral amino acids with side chains facing towards each other in the centre of the pore. The so called intermediate ring (constituting of αE241 and equivalent) and the adjacent to $\alpha$ E241 in helical configuration central ring, (formed by $\alpha$ L244 and equivalent) form a narrow constriction of the ion pore, hence have the strongest effect on the conductance rate [@imoto1991; @imoto1988]. In addition, the negatively charged side chains of intermediate ring are crucial for ion selectivity [@konno1991]. The gating of the channel is governed by conserved leucine residues, slightly towards the extracellular side from the centre of the bilayer with side chains projecting inwards [@unwin1995], hence occluding the passage for ions.
The stiochiometry 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. Constituting nAChR subunits are arranged pseudosymmetrically, around the central ion conduction pore [@brisson1985]. The subunit composition of the neuromuscular nAChR follows the strict order of $\alpha1$, $\beta1$, $\delta$, $\alpha1$, $\gamma$ (clockwise). Each subunit of the nAChR contains 4 transmembrane helices [@noda1982; @noda1983] named M1, M2, M3 and M4, as moving from N- to C- terminus. M1, M3 and M4 are exposed to the plasma membrane [@blanton1994], shielding M2, pore-forming helices [@imoto1986; @hucho1986] from the hydrophobic environment of the bilayer. As the outer helices progress from the outer to the inner leaflet of the membrane, they tilt inwards [@miyazawa2003], narrowing down the width of the channel. M2 on the other hand, bends roughly in the middle of the bilayer [@unwin1995], where it forms the most restricted part of the ion conductivity pathway. There are hydrophobic interactions between the outer helices, which stabilise the outer wall of the receptor and hence limit the conformational changes adopted by the inner helix. In contrast there are no extensive bonds between the inner and outer helices [@miyazawa2003]. As lining pore structures, the inner helix and flanking sequences contain molecular determinants for ion selectivity, permeability, the rate of conductance and gating. These were investigated by pharmacological, biochemical and electrophysiological approaches. [@imoto1988; @imoto1991; @konno1991] investigated the function of several rings of anionic and neutral amino acids with side chains facing towards each other in the centre of the pore. The so called intermediate ring (constituting of αE241 and equivalent) and the adjacent to $\alpha$ E241 in helical configuration central ring, (formed by $\alpha$ L244 and equivalent) form a narrow constriction of the ion pore, hence have the strongest effect on the conductance rate [@imoto1991; @imoto1988]. In addition, the negatively charged side chains of intermediate ring are crucial for ion selectivity [@konno1991]. The gating of the channel is governed by conserved leucine residues, slightly towards the extracellular side from the centre of the bilayer with side chains projecting inwards [@unwin1995], hence occluding the passage for ions.
<!-- ALSO TALK ABOUT THE NEGATIVELY CHARGED VESTIBULE HERE -->
(ref:structure-nachr) **Structural features of the nicotinic acetylcholine receptor.** Torpedo nAChR is a transmembrane protein, made up of 5 subunits (colour-coded), arranged around the ion conductivity pore. Each subunit consists of extracellular ligand-binding, transmembrane and intracellular domain (a) (PBD code:2BG9). Extracellular domain of a single subunit consists of 10 $\beta$-strands and N-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.
......@@ -231,32 +234,34 @@ The stechiometry and structural details of muscle type nAChRs were confirmed by
knitr::include_graphics("fig/general_intro/png/crystal_structure_nachr.png")
```
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 is has similar structure to the extracellular domain of the nAChRs mammalian $\alpha1$ [@dellisanti2007] and $alpha7$ [@li2011]. It is a homopentamer with N-terminal helix and 10 $\beta$sheets. It also shares similar pharmacological properties to this receptor. AChBP binds to classical nAChR agonist and antagonists: nicotine, acetylcholine and $\alpha$-bungarotoxin [@smit2001]. Therefore AChBP is considered a good model for the nAChR ligand-binding domain structural studies. The structures of AChBP inactive [@brejc2001], bound to agonist and antagonist [@celie2004; @hansen2005], chimera $\alpha1$ [@dellisanti2007] and $\alpha7$ are known [@li2011]. The common structural features of the ligand binding site emerge from all available data. Here data from Ls will be discussed.
### Model of the binding site
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 is has similar structure to the extracellular domain of the nAChRs mammalian $\alpha1$ [@dellisanti2007] and $alpha7$ [@li2011]. It is a homopentamer with N-terminal helix and 10 $\beta$sheets. It also shares similar pharmacological properties to this receptor. AChBP binds to classical nAChR agonist and antagonists: nicotine, acetylcholine and $\alpha$-bungarotoxin [@smit2001]. Therefore AChBP is considered a good model for the nAChR ligand-binding domain structural studies. The structures of AChBP inactive [@brejc2001], bound to agonist and antagonist [@celie2004; @hansen2005], chimera $\alpha1$ [@dellisanti2007] and $\alpha7$ are known [@li2011]. The common structural features of the ligand binding site emerge from all available data. Here data from the great pond snail *Lymnaea stagnalis* (Ls) will be discussed.
<!-- (it unlike Ac, all aromatic residues in Ls are conserved). -->
### Binding site
### Agonist binding site ###{#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 receptors, 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 originated from loop D (loop $\beta2$-$\beta3$), E (loop $\beta5$-$\beta6$) and F (loop $\beta8$-$\beta9$). Specific residues involved in the formation of the ligand binding pocket were depicted by the molluscan AChBP (Figure \@ref(fig:binding-pocket-label)). Amino acids of the principal component are: Tyr93, Trp147, Tyr188 and Tyr195, whereas non-$\alpha$ component contributes Trp53, Gln55, Arg104, Val106, Leu112 and Met114, Tyr164.
The nicotinic acetylcholine receptor binding pocket is formed on the interface of the adjacent subunits [@brejc2001; @middleton1991; @blount1989, Figure \@ref(fig:binding-pocket-label)]. In case of the neuromuscular heteropentameric receptors, it constitutes of $\alpha$ and non-$\alpha$ subunit contributions, whereas in homopentameric or $\alpha$ 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 originated from loop D (loop $\beta2$-$\beta3$), E (loop $\beta5$-$\beta6$) and F (loop $\beta8$-$\beta9$). Specific residues involved in the formation of the ligand binding pocket were depicted by the molluscan AChBP (Figure \@ref(fig:binding-pocket-label)). Amino acids of the principal component are: Tyr93, Trp147, Tyr188 and Tyr195, whereas non-$\alpha$ component contributes Trp53, Gln55, Arg104, Val106, Leu112 and Met114, Tyr164.
(ref:binding-pocket) **The ligand binding domain of acetylcholine binding protein.** Agonist binds to the loops situated in the adjecent subunits of the nAChR. In muscle type receptor, there are 2 binding sites, and thee are 5 in homopentameric receptor, such as mammalian $\alpha7$ (a). The ligand binding pocket of the AChBP (PDB:1I9B) is formed from loops of the 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.
(ref:binding-pocket) **The ligand binding domain of acetylcholine binding protein.** Agonist binds to the loops situated in the adjecent subunits of the nAChR. In muscle type receptor, there are 2 binding sites, and thee are 5 in homopentameric receptor (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")
```
### Pharmacophore
### Agonist pharmacophore ####{#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, $\alpha2$ extracellular domain bound to epibatidine [@kouvatsos2016] and $\alpha4\beta2$ receptor bound to nicotine [@morales-perez2016] have been obtained. These, together with the AChBP structures provide details of how structurally varied agonists bind to nAChRs.
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.
Agonist are buried on the interface of the neighboring subunits. They are stabilised in the binding pocket 5 conserved aromatic residues from A, B and C loops of the principal site (known as the aromatic box) engulf the cationic nitrogen of bound agonist. There are two major and conserved features: caption - $\pi$ interaction and hydrogen bond.
Agonist are 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 the 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 EC50 between 0.4 and 1.6 mM, whereas the EC50 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). Interactions with the receptor based on the crystal structure of the AChBP and nicotine (PB:1UW6) b. Chemical structures generated in ChemDraw. b taken from @blum2010. Cation-$\pi$ interactions between protonated nitrogen of tertiary amine and indole of TrpB.
(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). b is taken from @blum2010. Cation-$\pi$ interactions between protonated nitrogen of tertiary amine and indole of TrpB.
```{r pharmacophore-label, fig.cap="(ref:pharmacophore)", fig.scap='Nicotinic acetylcholine receptor agonist pharmacophore.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/nicotinic_interactions.png")
......@@ -264,26 +269,41 @@ knitr::include_graphics("fig/general_intro/png/nicotinic_interactions.png")
### Neonicotinoid-pharmacophore
Structure of AChBP proved to be valuable in determining structural elements which may account for neonicotinoids’ selectivity @ihara2008; @talley2008 derived crystal structures of the great pond snail (Lymnaea stagnalis, Ls) and California sea slug (Aplysia californica, Ac) AChBP complexed with bound neonicotinoids (imidacloprid, clothianidin, thiacloprid), and non-selective nAChR ligands- nicotinoids (nicotine, epibatidine and desmotroimidacloprid). Comparison of these structures revealed differences in binding modes between nicotinoids and neonicotinoids described below (see Appendix \@ref(fig:pharacophore-seq-label) for sequence alignment), which allowed for predictions of the binding interactions between neonicotinoids and insect receptors (Figure \@ref(fig:imi-binding-label)). The positioning of the pyridine ring of neonicotinoids and nicotinoids is virtually identical. In imidacloprid bound structure, nitrogen forms hydrogen bond with the amide group of Met114 and carbonyl group of Leu102 of loop E, via water molecule. The presence of water molecule, position of the nitrogen and similar interactions are also seen in nicotinic-bound structures [@celie2004; @ihara2008; @talley2008). In addition, chlorine atom of imidacloprid, thiacloprid as well as epibatidine makes van der Waals interactions with oxygen of Ile106 and oxygen of Met116 of AChBP.
Structure of AChBP proved to be valuable in determining structural elements which may account for neonicotinoids’ selectivity. @ihara2008; @talley2008; @ihara2014 derived crystal structures of the great pond snail (Lymnaea stagnalis, Ls) and California sea slug (Aplysia californica, Ac) AChBP complexed with bound neonicotinoids (imidacloprid, clothianidin, thiacloprid), and non-selective nAChR ligands- nicotinoids (nicotine, epibatidine and desmotroimidacloprid). Comparison of these structures revealed differences in binding modes between nicotinoids and neonicotinoids (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)).
One of the main features of nAChR-bound nicotine is the presence of the cation-$\pi$ interactions between the cationic nitrogen of the pyrrolidine ring and TrpB. In imidacloprid bound structures, the ring stacks with aromatic residues Tyr185 and Tyr188 of loop C. These stacking interactions result in the formation of CH-$\pi$ interactions between the methyline bridge (CH2-CH2) of imidacloprid and TrpB. The 5-member ring of imidacloprid stacks with evolutionary conserved tyrosine residue (Tyr188 of Ac and Tyr 185 of Ls), which is also seen in epidibine, but not in nicotine. All residues described so far are conserved in other agonist-bound nAChR structures, therefore do not account for neonicotinoids-selectivity.
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 Ls 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 identical. 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].
The differences come to light when one begins to dissect the interactions between imidacloprid ring substituents and the AChBP. Partially positive nitro group (NO2) bridges to glutamine of loop D (Gln55 in Ls and Glu 57 in As) via hydrogen bond. It is interesting that in some nAChR subunits, such as chicken $\alpha2$, honeybee $\alpha6$, $\alpha7$, $\beta1$ and $\beta2$, glutamine corresponds to basic residue (lysine/arginine). Basic residues electrostatically attract nitro group, possibly forming a hydrogen bond, which in turn would strengthen the stacking and aromatic CH/$\pi$ hydrogen bond interactions between the ring and the protein. In contrast, other subunits contain either acidic or polar amino acids in the exact position, repulsing or forming no electrostatic interactions with imidacloprid. Thus it has been proposed that the basic residue in loop D interacting with the nitro group of the imidazole ring is important in confirming neonicotinoid selectivity in insect nAChR subunits. This is supported by the genetic studies. Loop D arginine to threonine mutation naturally occurring in $\beta1$ subunit of peach aphid *Myzus persicae*, and cotton aphid *Aphis 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.
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), as well as 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 (this interaction is also seen in epibatidine-bound structures) [@ihara2008]. These stacking interactions result in the formation of CH-$\pi$ interactions between the methyline bridge (CH2-CH2) 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 basic residue of loop D, which may account for the selectivity of neonicotinoids to insect receptors, originates from the complementary site of the binding pocket, suggesting the complementary binding site is more important than the principal binding site in determination of the neonicotinoid selectivity. This supports the ideas that selectivity of nAChR agents is determined by the complementary subunit amino acids [@marotta2014]. Swapping of $\beta$ subunits in heteromeric receptor diminished cytisine activity on the receptor [@harpsoe2013] which became responsive to cytisine at sub-$\mu$M concentrations by a mutation in a single amino acid in the complementary binding pocket [@marotta2014]. The variation in this binding pocket residues gives rise to ligand binding specifities and pharmacological differences between various compounds and receptors. So for example, conserved Trp in loop D is essential for the high affinity binding of $\alpha$-Bgtx to Ac AChBP [@hansen2004].
The differences come to light when one begins to dissect the interactions between imidacloprid ring substituents and the AChBP. Partially positive nitro group (NO2) of imidacloprid bridges to glutamine of loop D (Gln55) via hydrogen bond. This interaction was also seen in thiacloprid bound AChBP and in the Gln55Arg mutant of AChBP bound to clothiandin [@ihara2014]. It is interesting that in some nAChR subunits, such as chicken $\alpha2$, honeybee $\alpha6$, $\alpha7$, $\beta1$ and $\beta2$, glutamine corresponds to basic residue (lysine/arginine). Basic residues electrostatically attract nitro group, possibly forming a hydrogen bond, which in turn would strengthen the stacking and aromatic CH/$\pi$ hydrogen bond interactions between the ring and the protein. In contrast, other subunits contain either acidic or polar amino acids in the exact position, repulsing or forming no electrostatic interactions with imidacloprid.
Basic residue in loop D is not the only feature determining the affinity of neonicotinoids to insect nAChRs. Amino acids in 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. Genetic analysis of imidacloprid-resistant strain of Nilaparvata lugens identified Y151S mutation in loop B of $\alpha1$ and $\alpha3$ [@liu2005]. This residue corresponds to LsAChBP H145 of the loop B, suggesting loop B may be also important.
(ref:imi-binding) **Residues forming interactions with nicotine and neonicotinoids in the binding site of AChBP**. Schematic representation of the agonist binding site of AChBP, highlighting residues interacting with nicotine and imidacloprid.
(ref:imi-binding) **The predicted interactions between imidacloprid and insect nAChR.** Electrostatic interactions between the nitro group of imidazole ring of imidacloprid and basic residue of Loop D strengthens the interactions between neonicotinoids and nAChRs. Imidazole ring forms stacking interactions with Tryptophan of loop B. Its position is also stabilised by the Tyrosines of loops A and C. Carbonyl group of TrpB can form alternative interaction with imidacloprid. Image taken and modified from @matsuda2005.
```{r imi-binding-label, fig.cap="(ref:imi-binding)", fig.scap= "Residues forming interactions with nicotine and neonicotinoids in the binding site of AChBP", 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 NO2 group of clothianidin and CN group of thiacloprid, but does not interact with imidalcoprid (Figure \@ref(fig:all-neonics-binding-label)) [@ihara2014].
```{r imi-binding-label, fig.cap="(ref:imi-binding)", fig.scap= "The predicted interactions between imidacloprid and insect nAChR", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/imi_binding_site.png")
(ref:all-neonics-binding) **Residues forming interactions with neonicotinoids in the binding site of AChBP**. Schematic representation of the agonist binding site of AChBP, 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="Residues forming interactions with neonicotinoids in the binding site of AChBP", fig.align='center', echo = FALSE, }
knitr::include_graphics("fig/general_intro/png/binding_all_neonics.png")
```
#### Neoniotinoid-selectivity
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 the genetic studies. Loop D arginine to threonine mutation naturally occurring in $\beta1$ subunit of peach aphid *Myzus persicae*, and cotton aphid *Aphis 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 equivalent of Gln55 and were mutated to basic residues showes increased binding affinity of thiacloprid and clothianidin, but not nicotine or acetylcholine [@ihara2014], providing further evidence that these residues are important in confering high binding affinity of neonicotinoids.
Genetic studies identified other amino acids with a potential importance in confering neonicotinoid-selectivity. Imidacloprid-resistant strain of *Nilaparvata 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.
### Cholinergic neurotransmission
<!-- Upon arrival of an electrical signal at the presynpatic terminal, acetylcholine is released into the synaptic cleft. It then binds to nicotinic acetylcholine receptors (nAChRs) expressed at the post-synpatic membrane. Binding of acetylcholine results in depolarisation and excitation of the post-synaptic neurons, or muscle contraction at the neuromusclular junction (NMJ) [@hille1978]. Acetylcholine can also act on other class of receptors, the metabotropic cholinergic G-protein coupled receptor, which are involved in the modulatation of neurotransmission release. The acetylcholine-evoked signal is terminated mainly by synaptic enzyme cholinesterase which hydrolyses acetylcholine to choline and acetate [@fukuto1990], but also by choline uptake to the presynaptic cell by Na^+^-choline transporter. -->
Cholinergic neurotransmission is the process of signal propagation between neurons as well as neurons and muscle cells mediated by a neurotransmitter acetylcholine (ACh) (Figure \@ref(fig:cholineric-synapse-label)) [@williamson2009], via nAChRs. 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].
......@@ -310,9 +330,9 @@ knitr::include_graphics("fig/general_intro/png/synapse_general_2.png")
<!-- $\alpha$-bungarotoxin ($\alpha$-bgtx), is a 74-amino acid long, 8 kDa proteins isolated from the venom of a snake *Bungarus multicinctus*. It binds with high affinity to the NMJ post-synaptic membranes [@lee1967] and blocks synaptic responses evoked by acetylcholine and other agonists [@chang1963] by blocking the access of an agoinist to the nAChR binging site [@@mishina1984]. -->
## Biological relevance of the cholinergic neurotransmission
## Biological relevance of the cholinergic neurotransmission ##{#insectachtransmission}
Acetylcholine is the main neurotransmitter in the nervous system of insects [@florey1963]. Its action is mediated predominately by nAChRs, which are the main cholinergic receptor type in their central nervous system [@breer1987]. The presence of nAChR in various brain regions has been detected using biochemical and electrophysiological techniques on neuronal preparations extracted from the Fruit fly *Drosophila melanogaster*, honey bee *Apis mellifera* and American cocroach *Periplaneia americana*. nAChRs have been found to be expressed in the regions associated with learning, formation of memory and the sensory processing [@heisenberg1998], namely the muschroom bodies [@kreissl1989; @gu2006; @oleskevich1999]. They are also present in the insect ganglia, which connects the brain to the peripheral nervous system. In particular, they were identified in the abdominal, thoracic and the terminal ganglia [@sattelle1981; @bai1992], which are involved in the movement of wings, abdomen and legs, as well as the regulation of the anal and reproductive muscles [@smarandache-wellmann2016]. In contrast to mammals and invertebrates (Table \@ref(chlinergic-nts)), insects do not express nAChRs at the neuromuscular junction.
Acetylcholine is the main neurotransmitter in the nervous system of insects [@florey1963]. Its action is mediated predominately by nAChRs, which are the main cholinergic receptor type in their central nervous system [@breer1987]. The presence of nAChR in various brain regions has been detected using biochemical and electrophysiological techniques on neuronal preparations extracted from the Fruit fly *Drosophila melanogaster*, honey bee *Apis mellifera* and American cocroach *Periplaneia americana*. nAChRs have been found to be expressed in the regions associated with learning, formation of memory and the sensory processing [@heisenberg1998], namely the muschroom bodies [@kreissl1989; @gu2006; @oleskevich1999]. They are also present in the insect ganglia, which connects the brain to the peripheral nervous system. In particular, they were identified in the abdominal, thoracic and the terminal ganglia [@sattelle1981; @bai1992], which are involved in the movement of wings, abdomen and legs, as well as the regulation of the anal and reproductive muscles [@smarandache-wellmann2016]. In contrast to mammals and invertebrates (Table \@ref(tab:chlinergic-nts)), insects do not express nAChRs at the neuromuscular junction.
### Biological role of nAChRs in insects
......@@ -322,21 +342,21 @@ Nicotine is a naturally occurring alkaloid found in the *Solanaceae* family of p
### Neonicotinoids target nAChRs ### {#neonicstarget}
#### Electrophysiological evidence
#### Electrophysiological evidence ####{#electrophysevidence}
The effects of neonicotinoids on the neuronal transmission was investigated on insect neuronal preparations which express high levels of nAChRs (Section \@ref()).
The effects of neonicotinoids on the neuronal transmission was investigated on insect neuronal preparations which express high levels of nAChRs.
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buchingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects. This conclusion was supported by the behavioural observation, whereby the neonicotinoid intoxication mirrors intoxication seen with cholinergic agents (Section . In response to imidacloprid, insects become hyper excited as evident by excessive pacing. They then collapse and exhibit diminishing uncoordinated leg and abdomen movement until eventual death [@sone1994; @elbart1997; @suchail2001]. Sub-lethal doses (i.e. < 4 nM) have distinct effect, such as an inhibition of feeding leading to starvation [@nauen1995; @elbart1997].
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects. This conclusion was supported by the behavioural observation, whereby the neonicotinoid intoxication mirrors intoxication seen with cholinergic agents (Section . In response to imidacloprid, insects become hyper excited as evident by excessive pacing. They then collapse and exhibit diminishing uncoordinated leg and abdomen movement until eventual death [@sone1994; @elbart1997; @suchail2001]. Sub-lethal doses (i.e. < 4 nM) have distinct effect, such as an inhibition of feeding leading to starvation [@nauen1995; @elbart1997].
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured cocroach [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons. These data provide evidence that neonicotinoids act directly on the post-synaptic neuron in both target and non-target insects.
Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC50 values (concentrations at which the half of the maximum current was observed) are in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(otentpests)). 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.
Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC50 values (concentrations at which the half of the maximum current was observed) are in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(potentpests)). Neonicotinoid-induced currents were reminiscent of those induced by acetylcholine and nicotine, and were prevented by the application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine), suggesting neonicotinoid-induced currents are due to the activation of nicotinic receptors.
#### Biochemical evidence
@tomizawa1996 developed neonicotinoid agarose affinity column to isolate proteins with high binding affinity to neonicotinoids. Using Drosophila and Musca head membrane preparations, he identified three nAChR subunits as potential neonicotinoid-targets.
##### Ligand binding studies
##### Ligand binding studies #####{#ligbinding}
The binding affinity of imidacloprid to nAChRs expressed in insect membrane homogenates was assessed in the saturation ligand binding studies.
......@@ -371,11 +391,11 @@ In addition to the saturation studies, the competitive ligand binding studies we
Various concentrations of neonicotinoid prototype isothiaocynate were incubated with the homogenate of fruit fly *Drosophila melanogaster* and a homogenate of the abdominal nerve cords of *Periplaneta americana* before the exposure to radiolabelled nAChR antagonist $\alpha$-bgtx [@gepner1978]. Isothiaocynate inhibited binding of $\alpha$-bgtx in the concentration dependent manner [@gepner1978], suggesting the two compounds share the binding site. Similarly, imidacloprid has been shown to displace $\alpha$-bgtx from brain membrane preparations from honey bee *Apis mellifera* [@tomizawa1992; @tomizawa1993], *Drosophila melanogaster* [@zhang2004], house fly *Musca domestica* and isolated cockroach nerve cords [@bai1991].
#### Genetic evidence
#### Genetic evidence ####{#resgenevidence}
Resistance to neonicotinoids arises from mutations in nAChR subunits. Field isolates of peach aphid *Myzus persicae* [@bass2011], the cotton aphid *Aphis gossypii* [@hirata2015; @hirata2017] and the Colorado potato beetle *Leptinotarsa decemlineata* [@szendrei2012], as well as lab-isolates of brown planthopper, *Nilaparvata lugens* [@liu2005], fruit fly *Drosophila melanogaster* [@perry2008] with decreased sensitivity to neonicotinoids have been identified. Behavioral analysis shows that their sensitivity is up to 1500-fold lower in comparison to the reference strains, as shown by the shift in LD50. Analysis of the coding genome of the resistant strains identified mutations in nAChR subunit coding sequence [@bass2011; @perry2008; @hirata2015].
### Mode of action of neonicotinoids
### Mode of action of neonicotinoids ###{#moaneonicsinsects}
Neonicotinoids can have diverse mode of action. The currents produced by neonicotinoids and ACh on cultured or isolated insect neuronal preparation were compared. Neonicotinoids evoking current lower than that evoked by ACh were classed as partial agonists, those eliciting similar response were classed as true agonists, whereas those more efficacious than ACh, super-agonists. Thiacloprid and imidacloprid are partial agonists, nitenpyram, clothianidin, acetamiprid and dinotefuran are true agonists, whereas thiamethoxam has no effect on the isolated American cockroach thoracic ganglion neurons [@tan2007]. This differs from the mode of action of neonicotinoids on cultured terminal abdominal ganglion neurons of this insect. Currents produced by all neonicotinoids tested was lower than that evoked by ACh [@ihara2006], suggesting they are all partial agonists on these cells. The mode of action of neonicotinoids on the fruit fly [@brown2006] and honey bee neurons [@palmer2013] differs still, implying the presence of distinct nAChRs in different insect species and neuronal preparations.
......@@ -426,13 +446,13 @@ library(kableExtra)
threeparttable = T)
```
### nAChR subunits in insects ###{expressionfail}
### nAChR subunits in insects ###{#expressionfail}
nAChR are assemblies of 5 different or identical receptor subunits (Section \@ref(structure)). Each subunit is encoded by a separate gene and is classified as either $\alpha$ or non-$\alpha$, depending on the primary amino acid sequence, whereby $\alpha$ subunits contain a disulphide bond formed between the adjacent cysteines in the ligand binding domain (Figure \@ref(fig:structure-nachr-label)). Genome sequencing projects enabled identification of nAChR subunit families in several insect species. Fruit fly and model organism *Drosophila melanogaster* has 10 subunits, 7 of which are $\alpha$ ($\alpha1-7$) and 3 are $\beta$ ($\beta1-3$) [@adams2000a; @sattelle2005]. There are 11 subunits in the beneficial insect honeybee *A. mellifera* ($\alpha1-9$, $\beta1-2$) [@jones2006a; @consortium2006], 12 subunits in the pest red flour beetle *Tribolium castaneum* ($\alpha1-11$,, $\beta1$) [@consortium2008] and 7 in the Pea Aphid, *Acyrthosiphon pisum* ($\alpha1-6$, $\beta1-2$) [@yi-peng2013; @Consortium2010]. With the aid of molecular cloning techniques, equivalent subunits have been identified in many other insects, including cat flea *Ctenocephalides felis* [@bass2006] and green peach aphid *Myzus persicae* [@huang2000]. Amino acid sequence alignment of equivalent subunits revealed that they are highly conserved, with sequence identity typically greater than 60 % [@jones2010].
Insect nAChR gene families are among the least diverse when compared to other animal phyla. Mammals express 17 subunits: $\alpha1-10$, $\beta1-4$, $\delta$, $\gamma$ and $\epsilon$ [@millar2009] and there are 29 subunits in the representative of the phylum *Nematoda, C. elegans* [@jones2007b].
### RIC-3 improves recombinant nAChR assembly ###{ric3insect}
### RIC-3 improves recombinant nAChR assembly ###{#ric3insect}
<!-- There are different receptor types in insects. -->
<!-- https://radar.brookes.ac.uk/radar/file/c59cbdb5-d171-49e0-b0e4-101c261c72ed/1/fulltext.pdf -->
......@@ -460,7 +480,7 @@ knitr::include_graphics("fig/general_intro/png/nAChR_turnover.png")
Difficulties in expression of recombinant insect nAChRs (Section \@ref(expressionfail)) hiders their pharmacological analysis and identification of receptors sensitive to neonicotinoids. Insect-mammal hybrid receptors served as a platform to investigate the potency and affinity of neonicotinoids.
#### High affinity of neonicotinoids to insect-chimera receptors
#### High affinity of neonicotinoids to insect-chimera receptors ####{#chimerareceptors}
Mammalian $\alpha4$/$\beta2$ receptor expresses well in Xenopus oocytes [@cooper1991] and cell lines [@lansdell2000] and it has low affinity to imidacloprid (Kd >1000 $\mu$M) [@lansdell2000]. $\beta2$ from rat and chicken has been shown to enable recombinant expression of several insect $\alpha$ subunits in cell lines. Chimera of rat $\beta2$ and $\alpha$ subunits from the fruit fly *Drosophila melanogaster* [@lansdell2000], aphid *Myzus Persicae* [@huang1999], planthopper *Nilaparvata lugens* [@liu2009], cat flea *Ctenocephalides felis* [@bass2006] and sheep blowfly *Lucilia cuprina* [@dederer2011] have been generated. It needs to be noted that the potency of neonicotinoids on these receptors is unknown due to the lack of reported data, suggesting these receptors are not functional. However, their pharmacological profiles have been determined using saturation ligand binding studies [@hulme2010] (Table \@ref(tab:bindignrecombinant)).
......@@ -558,7 +578,7 @@ Subunits with various degree of sensitivity to neonicotinoids, suggesting some a
### 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(resistancegenintro)). In addition, by expressing nAChRs from pest and other species identification of compounds with no adverse effects on beneficial insects and other biologically important species may be achieved. Model organism *C. elegans* is a system in which the mode of action and the selective toxicity can be studied.
Recombinant insect nAChR are notoriously difficult to express. Several interventions have been tested including expression of hybrid receptor in which insect subunits have been co-expressed with mammalian ones. It need to be noted that this method has several limitations. First, hybrid receptors are not biologically relevant, thus conclusions from these studies should be drawn with caution. Second, some some of the expressed receptors may be folded, but not functional. Lastly, this method enabled expression of only a handful of receptors, thus most remained uncharacterised. This hinders their pharmacological characterisation and identification of subunits important in conferring the agricultural role of neonicotinoids. Heterologous expression of nAChR from insects and other species would allow for the characterisation of the interactions of these proteins with neonicotinoids to better define their mode of action and selective toxicity. Development of the platform in which the heterologous expression of insect nAChRs could be achieved, would open the door to screening of novel insecticides, to combat emerging and spreading neonicotinoid-resistance (Section \@ref(resgenevidence)) and @charaabi2018). In addition, by expressing nAChRs from pest and other species identification of compounds with no adverse effects on beneficial insects and other biologically important species may be achieved. Model organism *C. elegans* is a system in which the mode of action and the selective toxicity can be studied.
## Overview of *C. elegans*
......@@ -608,7 +628,7 @@ Many of the *C. elegans* behaviours are regulated by acetylcholine, which is the
### Evidence from behavioural analysis
The synthesis and packing of acetylcholine into synaptic vesicles are essential steps in cholinergic neurotransmission. These functions are mediated by several proteins (Figure \@ref(fig:cholsynapsecelegand)). Choline acetyltransferase (ChAT) encoded by the cha-1 gene catalyses the formation of acetylcholine [@rand1985]. Vesicular acetylcholine transferase (VAChT) encoded by unc-17 loads acetylcholine into synaptic vesicles [@alfonso1993]. Null mutations of these genes are lethal due to the inhibition of worm's locomotion and feeding and its eventual death due to starvation [@rand1989; @alfonso1993]. Polymorphic ChAT and VAChT mutants in which the expression is reduced, but not abolished, revealed somewhat opposite phenotype. The pharyngeal pumping both in the presence and absence of food was reduced [@dalliere2015] the movement highly uncoordinated and jerky [@rand1984], whereas egg-laying increased [@bany2003].
The synthesis and packing of acetylcholine into synaptic vesicles are essential steps in cholinergic neurotransmission. These functions are mediated by several proteins (Figure \@ref(fig:cholsynapsecelegand-label)). Choline acetyltransferase (ChAT) encoded by the cha-1 gene catalyses the formation of acetylcholine [@rand1985]. Vesicular acetylcholine transferase (VAChT) encoded by unc-17 loads acetylcholine into synaptic vesicles [@alfonso1993]. Null mutations of these genes are lethal due to the inhibition of worm's locomotion and feeding and its eventual death due to starvation [@rand1989; @alfonso1993]. Polymorphic ChAT and VAChT mutants in which the expression is reduced, but not abolished, revealed somewhat opposite phenotype. The pharyngeal pumping both in the presence and absence of food was reduced [@dalliere2015] the movement highly uncoordinated and jerky [@rand1984], whereas egg-laying increased [@bany2003].
### Pharmacological evidence
......@@ -630,34 +650,47 @@ Aldicarb is a synthetic carbamate mainly used as a nematicide (compound used to
(ref:cholsynapsecelegand) **Enzymes and transporters at the *C. elegans* cholinergic synapse.** Upon release into the synaptic cleft, acetylcholine is broken down to choline and acetate by acetylcholinesterase (AChE). Choline is taken up to the pre-synapse by a choline transporter (ChT). The acetyl group in transferred onto choline to product acetylcholine; a reaction catalysed by choline transferase (ChAT). Generated acetylcholine is pumped back into the synaptic vesicle by the vesicular acetylcholine transporter (AChT) for re-cycling. Names of genes are depicted in small blue letters. Image taken from @rand2006.
```{r cholsynapsecelegand-label, fig.cap="(fig:cholsynapsecelegand)", echo=FALSE, fig.scap='Enzymes and transporters at the *C. elegans* cholinergic synapse.',fig.align='center', echo = FALSE}
```{r cholsynapsecelegand-label, fig.cap="(ref:cholsynapsecelegand)", echo=FALSE, fig.scap='Enzymes and transporters at the *C. elegans* cholinergic synapse.',fig.align='center', echo = FALSE}
knitr::include_graphics("fig/general_intro/png/cholinergic_synapse_C.elegans.jpg")
```
### nAChRs
The action of acetylcholine is mediated by nAChR. *C. elegans* contains 29 genes encoding for nAChR subunits [@jones2007b], 22 of which are $\alpha$-subunits (Table \@ref(tab:celegans-nachrs)). The receptor subunits are assigned to five groups based on the sequence homology: DEG-3, ACR-16, ACR-8, UNC-38, and UNC-26. The nAChRs are expresses at the neuromuscular junction [@richmond1999] and in the nervous system [@lewis1987].
The action of acetylcholine is mediated by nAChR. *C. elegans* contains 29 genes encoding for nAChR subunits [@jones2007b]. The receptor subunits are assigned to five groups based on the sequence homology: DEG-3, ACR-16, ACR-8, UNC-38, and UNC-26 Figure (\@ref(fig:seqidentityecd-label)).
To date, four receptors 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 body wall muscle differentiated based on their pharmacology into L-(levamisole)type and N-(nicotine)-type [@richmond1999]. The subunit composition of these receptors is respectively: UNC-29, UNC-38, UNC-63, LEV-1, LEV-8 associated with auxiliary subunits RIC-3, UNC-50, and UNC-74 [@boulin2008] and ACR-16 homopentamer [@touroutine2005] (more details is Section @\ref(muscletypenachr)). EAT-2 is a predicted $\beta$ nAChR subunit expressed in the pharyngeal muscle, believed to assemble with auxilary subunit EAT-18, based on common localisation and behavioural phenotypes of *eat-2* and *eat-18 C. elegans* mutants [@mckay2004] (Section @\ref()). Based on the expression in Xenopus oocytes, ACR-2 and UNC-38 may co-assembly [@squire1995]. However the levamisole-induced currents were of low amplitude, which may suggest a necessity for auxiliary subunits.
Sequence identity between the insect and *C. elegans* subunits is low. Mean identity is 35 %. Least homologous are members of the DEG-3 family with the mean value of 28 %. the other three groups between 37 and 41 %.
```{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)
Low similarity between the residues of the ligand binding domain suggest these subunits diverged during the evolution thus have distinct pharmacophore.
The nAChRs are expresses at the neuromuscular junction [@richmond1999] and in the nervous system [@lewis1987].
To date, four receptors 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 body wall muscle differentiated based on their pharmacology into L-(levamisole)type and N-(nicotine)-type [@richmond1999]. The subunit composition of these receptors is respectively: UNC-29, UNC-38, UNC-63, LEV-1, LEV-8 associated with auxiliary subunits RIC-3, UNC-50, and UNC-74 [@boulin2008] and ACR-16 homopentamer [@touroutine2005] (more details is Section \@ref(muscletypenachr)). EAT-2 is a predicted $\beta$ nAChR subunit expressed in the pharyngeal muscle, believed to assemble with auxilary subunit EAT-18, based on common localisation and behavioural phenotypes of *eat-2* and *eat-18 C. elegans* mutants [@mckay2004]. Based on the expression in Xenopus oocytes, ACR-2 and UNC-38 may co-assembly [@squire1995]. However the levamisole-induced currents were of low amplitude, which may suggest a necessity for auxiliary subunits.
<!-- # ```{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) -->
<!-- # ``` -->
(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 *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")
```
## Heterologous expression of nAChRs in *C. elegans*
## Heterologous expression of nAChRs in *C. elegans* ##{hetexpeffectsofphysiology}
*C. elegans* can be used as a platform to study functional and pharmacological properties of nAChRs. This method relays on the ability to generate transgenic worms by microinjection.
......
</
---
nocite: |
@chen1997, @araujo1988, @couturier1990, @cooper1991, @lee1967, @brown1936, @mishina1986, @zirger2003, @mongeon2011, @lewis1987, @treinin1998, @richmond1999, @boulin2008, @touroutine2005,
...
# General introduction {#generalintro}
## Chemical treatment in agriculture
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]. Their identity evaluated over the years to improve the effectiveness and reduce the undesirable effects on human health and the environment [@casida1998].
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, has been used in France, England and the US 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 use 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. DTT 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]. DTT 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 pyrothroids (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 bn Euros, 1.19 bn Euro and 1.169 bn Euro, respectively [@jeschke2011]. The main issue associated with the use of organophosphates and carbamates is their ability to cause serious human poisonings, some of 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") %>%
add_footnote("Sales as of 2008, according to Jeschke et al. 2011",
threeparttable = T)
```
## Neonicotinoids
### Synthesis
In 1970s, the scientists of Shell Development Company Biological Research Centre in California identified alpha- DBPN (2-(dibromonitromethyl)-3-(methylpyridine)), first synthesised by Prof. Henry Feuer [@feuer1986]. This lead compound showed low insecticidal activity against aphid and house fly [@tomizawa2003; @tomizawa2005]. Structural alterations of DBPN resulted in production of nithiazine (Figure \@ref(fig:neonics-structure-label)). Nithiazine showed improved insecticidal activity and was particularly effective as a new housefly repellent [@kollmeyer1999]. Further replacement of the thiazine ring by chloropyridinylmethyl (CPM) group, addition of the imidazolidine or its acyclic counterpart, and retention of the nitromethylene group resulted in generation of more potent compounds, one of which, nitenpyram, exhibited particularly high efficacy. Regrettably, both nithiazine and nitenpyram are not useful in fields, as they are unstable in light. The latter however is successfully used in veterinary medicine as an external parasite treatment for cats and dogs.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
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 synthetic insecticides neonicotinoids.** Systematic modification of the lead and prototype compounds led to the discovery of seven neonicotinoids currently used in agriculture and animal health. They are 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 ###{#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 bn dollars [@jeschke2011]. This increased to 3.1 bn dollars in 2012 [@bass2015].
The widespread usage and monetary value of neonicotinoids is a reflection of their many advantages.
<!-- Important in the pest managment, used in over 120 coutries on 140 crop types [@jeschke2011]. -->
### Properties ##{#physchem}
One of the major benefits of neonicotinoids are their physical and chemical profiles (Table \@ref(tab:properties)).
#### Diverse methods of applications
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 can be 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 garden for flowers and vegetables and in agriculture on soft fruits and greenhouse crops. Low lipophilicity, indicated by octanol/water partition coefficient value (log Pow), suggest they do not bio-accumulate in the adipose tissues of animals [@turaga2016]. However, moderate water solubility combined with low lipophilicity means they may have a potential to accumulate in water.
<!-- Although structurally related nicotine has similar properties, it is not appropriate for the agricultural use due to low toxicity to insects [@nauen1996]. -->
```{r properties, echo=FALSE, warning = FALSE, message=FALSE}
library(kableExtra)
library(dplyr)
properties <- data.frame(
Compound = c("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 = 'Physichochemical properties of neonicotinoids',
) %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>%
add_footnote("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)
```
#### 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, as measured by the LC50/LD50 (the concentration/dose of a compound that kills 50 % of the population) in the acute toxicity assays (Table \@ref(tab:toxallanimal)). The lower the LC(D)50, the greater the potency of a compound.
Neonicotinoids are effective against a wide range of piercing-sucking pests such as cotton and peach aphids (*Aphis gossypii* and *Myzus persicae*) [@nauen1996; @mota-sanchez2006; @bass2011], domestic (*Malus domestica*) and may-flies (*Epeorus longimanus*) [@tomizawa2000; @alexander2007] as well as planthoppers (*Nilaparvata lugens*) [@zewen2003]. Their IC50 is in generally 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* 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. LC50s are lower upon systemic or oral administration in comparison to the topical exposure [@alexander2007]. Imidacloprid injected into the abdomen of American cockroaches *Periplaneta 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 *Myzus 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", "Thx", "Clo", "Clo", "Clo", "Clo", "Clo", "Clo", "Imi", "Imi", "Clo", "Clo", "Clo", "Imi", "Acet", "Nit", "Clo", "Thia", "Thia"),
Species = c("A. gossypii", "A. gossypii", "A. gossypii", "M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae", "M.persicae","M.persicae", "M.persicae", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "L. decemlineata", "N.lugens","M. domestica", "M. domestica", "E. longimanus", "E. longimanus", "A. mellifera", "A. mellifera", "A. mellifera","A. mellifera","A. mellifera", "Bobwhite quail", "Bobwhite quail", "Mallard duck", "Mouse", "O. mykiss", "L. macrochirus", " E. fetida", "E. fetida", "E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", " E. fetida", "M. incognita"),
Taxon = c("Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect","Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "Insect", "insect", "Insect", "Insect", "Bird", "Bird", "Bird", "Mammal", "Fish", "Fish", "Earth worm", "Earth worm","Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Earth worm", "Nematode"),
LD50 = c("-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "-", "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 (acute)", ">5040 mg/kg (5 days)", ">5230 mg/kg (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 (24 hrs)", "2.54 nM (96 hrs)", "6.88 $\\mu$M", "-", "-","-","-","-", "-", "-", "-", "424.51 $\\mu$M", "468.60 $\\mu$M", "4.81 $\\mu$M (24 hours)", "2.74 $\\mu$M (48 hours)", "62.08 $\\mu$M (14 days)", "24.24 $\\mu$M (7 days)", "24.27 $\\mu$M (14 days)", "11.93 $\\mu$M (14 days)", "12.08 $\\mu$M (14 days)", "26.75 $\\mu$M (14 days)", "3.72 $\\mu$M (14 days)", "10.60 $\\mu$M (14 days)", "143. 24 $\\mu$M (6 hours)"),
Bioassay = c("Topical", "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 %>%
kable("latex", align = "l", escape = F, booktabs = TRUE, longtable = TRUE,
caption = 'Toxicity of nicotine and neonicotinoids') %>%
kable_styling(font_size=10, position = "center", full_width = FALSE, latex_options = c( "hold_position", "repeat_header")) %>%
footnote(general = footnotea,
threeparttable = TRUE)
```
```{r tox, echo=FALSE, warning = FALSE, message = FALSE}
library(kableExtra)
library(dplyr)
# toxic <-data.frame (
# Drug = c("Thia", "Clo", "Nit", "Thia", "Clo", "Nit", "Nic"),
# Species = c("Aphis gossypii", "Aphis gossypii", "Aphis gossypii", "Aphis gossypii", "L. decemlineata", "L. decemlineata", "L. decemlineata"),
# Taxon = c("Insect", "Insect", "Insect", "Insect","Insect", "Insect", "Insect"),
# LD50 = c("", "", "", "0.18 ng/mg", "0.15 ng/mg", "0.20 ng/mg", "0.35 ng/mg"),
# LC50 = c("9.35 $\\mu$M", "7.29 $\\mu$M", "9.12 $\\mu$M", "", "", "", ""),