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nicotinic Acetylcholine Receptor α1: Biological Overview | References

BIOLOGICAL OVERVIEW

Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. This study used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. This is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance (Somers, 2017).

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor subfamily of ligand-gated ion channels (LGICs) that mediate the transduction of a chemical signal into an electrical signal. Activation in response to acetylcholine (ACh), the major excitatory neurotransmitter of the Drosophila melanogaster central nervous system, is on a micro- to submicrosecond timescale. nAChRs are expressed in a wide range of tissues, including but not limited to the nervous system. When expressed presynaptically, nAChRs can enhance neurotransmitter release, while postsynaptic expression mediates excitation. Like all LGICs, nAChRs are pentameric and can consist of several different subunits forming multiple receptor subtypes that vary in both their sensitivity to particular ligands and their permeability to particular cations. Individual subunits consist of a pore-forming transmembrane domain coupled to a large extracellular N-terminal domain, which forms the endogenous ligand-binding site with adjacent subunits. Upon ACh binding, a conformational change occurs opening the channel pore to permit the flow of cations (Somers, 2017).

D. melanogaster has 10 nAChR subunits; however, only three of these have been associated with behavioral phenotypes, with specific roles described for Dα7 in the escape response and for both Dα3 and Dα4 in sleep behavior. Mutations in three D. melanogaster nAChR subunits confer resistance to two important classes of insecticides; Dα1 and Dβ2 to neonicotinoids, and Dα6 to spinosyns. Modification of an insecticide target protein can decrease the efficacy of an insecticide through altered affinity or pharmacological response. Modifications of this nature can provide a significant fitness boost to individuals in a population during insecticide treatment periods and can rapidly increase in frequency leading to control failures. However, allele frequencies will also be shaped by fitness costs if resistant mutations negatively impact reproductive output, either by reducing viability or the capacity to mate. This could be particularly true for alleles that directly modify the neonicotinoid-binding site as neonicotinoids occupy the same binding site as ACh. Loss-of-function mutants for the Dα1, Dα6, and Dβ2 genes are insecticide-resistant and viable. The viability phenotype suggests a level of functional redundancy among nAChR subunits. Questions remain as to whether individual subunits have other, nonredundant functions in controlling behavior or if the subtle pharmacological and physiological differences from compensating subunits manifests in altered behaviors. Impacts on mating behavior are of particular interest with respect to potential fitness costs (Somers, 2017).

While invertebrate nAChR pharmacology and biochemistry with respect to insecticide binding has been examined in detail, little research has been devoted to the endogenous functions of these receptors. D. melanogaster is commonly used as a model insect system to study many facets of biology, including insecticide resistance. A large number of well-defined behavioral paradigms exist to investigate the roles of genes in traits, including sleep and cognition through to mating and auditory faculties (Somers, 2017).

This study reports the creation of a Dα1 knockout mutant and a rescue system through transgene expression. This provided a consistent genetic background to analyze several behavioral paradigms. These reagents were used to identify roles for the D. melanogaster Dα1 subunit in mating, locomotion, and sleep, demonstrating the diverse pleiotropic influences that nAChRs can have on insect behavior. This research has relevance to the consideration of fitness costs that might be associated with resistance conferring mutations in Dα1 orthologs in pest insects, and the behavioral impact that exposure to neonicotinoids may have in beneficial species such as the western honey bee, Apis mellifera (Somers, 2017).

To generate a Dα1 null mutant, a modified ends-out targeting scheme was used. A precise 57-kb deletion of the Dα1 genomic region was created, then validated by Southern blot and sequencing. This mutant is referred to as Dα1^KO. Given that previously created Dα1 alleles are resistant to neonicotinoids (Perry, 2008), Dα1^KO was screened on two different neonicotinoids (imidacloprid and nitenpyram) and was found to be highly resistant to both. Calculated LC50 values and resistance ratios were consistent with those measured for Dα1 mutants studied previously (Perry, 2008). Dα1 and orthologs in pest species are well-established targets for several different neonicotinoid insecticides. Heterologous expression and affinity chromatography studies have also implicated Dα1 in directly binding imidacloprid at a site that overlaps that normally occupied by the ligand, ACh. Therefore, these resistance data matched the prediction that a mutant with a genomic deletion of Dα1 would be resistant to neonicotinoid insecticides (Somers, 2017).

The GAL4-UAS system was employed to create a phenotypic rescue in which a Dα1 cDNA clone was expressed in the Dα1^KO background. Expression of the Dα1 clone using the pan-neuronal elav::GAL4 driver rescued sensitivity to both imidacloprid and nitenpyram. The reversion of the resistance phenotype indicates that the subunit expressed from the Dα1 transgene is assembling into functional nAChRs that bind these insecticides (Somers, 2017).

The loss of Dα1 results in significant levels of resistance to neonicotinoids. However, the level of resistance is not of the same magnitude observed in the Dα6 knockout mutant, which is over 1000-fold resistant to spinosad. The Dα1^KO mutant is still susceptible when exposed to a high enough dose, which may be due to expression of other neonicotinoid-sensitive nAChR subtypes. Mutations in the orthologs of Dα3 and Dβ1 have been identified in neonicotinoid-resistant strains of Nilaparvata lugens and Myzus persicae, respectively. While only one imidacloprid-binding site has been reported in adult D. melanogaster and other Dipteran and Lepidopteran species, multiple binding sites have been reported in several Hemipteran species. Unlike Hemipterans, Dipterans and Lepidopterans are holometabolous insects that undergo complete metamorphosis from larva to adult. It is possible that as yet undescribed imidacloprid-sensitive nAChR subtypes are expressed in the larval life stages. Another possibility is that a novel subtype is formed as a consequence of the loss of the Dα1 subunit that alters the mutant's sensitivity to neonicotinoids (Somers, 2017).

RNAi knockdown of the Dα1 subunit resulted in defects in courtship and copulation behavior. Previous microarray studies, confirmed by RT-PCR, highlighted expression of Dα1 in both neuronal and reproductive tissues. Taken together, these lines of evidence suggested the potential for Dα1 to function in mating behavior (Somers, 2017).

Mating behavior can be influenced by genetic background, for example expression of w is important for visual cues and misexpression of this gene can trigger male-male courtship. As the Dα1^KO line was generated in the w¹¹¹⁸ background, both the mutant and the control line have functionally null copies of w. Therefore, the w⁺ X chromosome from another isogenic line, RAL059, was used to replace the w¹¹¹⁸ X chromosome present in both the Dα1^KO mutant line and the w¹¹¹⁸ background line. These lines will be referred to as the mutant and wild-type lines respectively (Somers, 2017).

Courtship behavior was measured in terms of the latency of courtship initiation by males after female introduction into the mating chamber. Flies that failed to initiate courtship within the allowed 10-min period were given a maximum value. Wild-type males initiated courtship in every trial, regardless of the genotype of the female partner. Mutant males only initiated courtship 65% of the time with wild-type females and 79% of the time with mutant females. Mutant males also took significantly longer to initiate courtship than wild-type males when paired with either wild-type or mutant females (Somers, 2017).

Copulation latency was also measured for the same flies. Mutant males rarely copulated within the 10-min period, only 15% of trials when paired with a wild-type female and only 3% when paired with a mutant female. Wild-type males were more successful, initiating copulation in 90% of the trials when paired with a wild-type female and in 56% of the trials when paired with a mutant female. No significant difference was observed in copulation latency of mutant males when paired with either wild-type or mutant females. In contrast, wild-type males did initiate copulation significantly faster with wild-type females compared to mutant females. This suggests that, unlike the latency of courtship initiation that is primarily influenced by the genotype of the male, both sexes contribute to the latency of copulation initiation (Somers, 2017).

The rescue system was again employed to see if expression of a Dα1 transgene could rescue the courtship and copulation phenotypes observed for the Dα1^KO mutant. Appropriate driver-only and UAS-only control flies were used for comparison to rescue flies. No discernible differences in courtship initiation were observed when wild-type males were crossed to control or rescue female flies. However, when crossed to wild-type female flies, male rescue flies were more successful in initiating courtship than male control flies. Male rescue flies also showed a significant decrease in courtship initiation when crossed to female wild-type compared to male control flies. Significant rescue of copulation initiation was also observed in both female and male rescue flies. Rescue flies were both more successful and faster at initiating copulation than the appropriate controls (Somers, 2017).

It is clear from the data that Dα1 plays a role in Drosophila mating behavior; however, the underlying mechanism remains unknown. Defects in ACh signaling have previously been associated with abnormal mating behavior. Analysis of mosaic mutants, defective for cholinergic signaling, identified a neuropile in the mushroom body calyx critical for normal male courtship behavior. Male-specific, cholinergic neurons have also been identified in the abdominal ganglion, the disruption of which significantly decreased male fertility, potentially due to their innervation of the male reproductive system. The disruption of these neurons has been hypothesized to result in the uncoordinated or altered release of sperm, seminal fluid, and accessory proteins. The study, performed by Acebes (2004) used the presynaptic choline acetyltransferase marker to identify these neurons; however, the receptors receiving this signal were not identified. The data from mating experiments and expression analysis suggests that Dα1 may be one of the specific nAChR subunits expressed in this pathway. Another possibility to consider is a higher processing role for Dα1 in mating behavior. The phenotypes observed in the Dα1^KO mutant are consistent with a defect in one or more sensory modalities. While there are specialized receptors responsible for detecting sensory stimuli, cholinergic signaling has been identified in connecting sensory circuitry to processing centers in the brain. Dα1 expression has previously been observed in the mushroom body calyx and lateral protocerebrum, which includes the lateral horn. Recently, the role of the lateral horn in locusts has been proposed to serve as a site for multimodal sensory integration. This may suggest that Dα1 plays a role in integrating multimodal courtship circuitry to higher sensory processing centers. Challenging Dα1^KO mutants with sensory-specific behavioral paradigms and neuron-specific rescue could test this hypothesis (Somers, 2017).

The possibility was explored that impaired locomotion may be contributing to the mating phenotype observed in the Dα1^KO mutant. Over a 24-hr period, the Dα1^KO mutant exhibited hyperactivity; however, it moved at a slower average speed. When average speed was binned in 3-hr intervals, the difference was significant during the middle two 3-hr bins of the day and the last three 3-hr bins of the night. Most importantly, there was no significant difference in average speed during the first 3-hr bin of the day, when courtship assays were performed, indicating that general locomotion deficits did not impact the measurement of mating behavior (Somers, 2017).

The Dα1^KO mutant also has an unusual pattern of sleep. Although there were no significant differences observed in total amount of sleep, mutant flies slept significantly less during the night, experiencing less sleep episodes of significantly shorter duration than wild-type controls. The total time of day sleep experienced by the mutant was significantly longer with a higher number of sleep episodes; however, there was no observable difference in episode length. Expression of the Dα1 transgene in the mutant background increased amount of sleep, both during the day and night. This increase was a result of longer sleep episodes, which may explain why both mutant and rescue flies both show less night sleep episodes. Rescue flies have less night sleep episodes due to the length of these episodes, whereas mutant flies may have trouble with sleep initiation and maintenance. The exceptional increase in episode length observed in rescue flies is likely due to nonnative expression of the transgene; however, it is clear Dα1 influences sleep (Somers, 2017).

While activation of all cholinergic neurons inhibits sleep in flies, different neuronal groups can be wake-promoting or sleep-promoting. Individual nAChR subunits also appear to have different roles in sleep regulation. From this study, Dα1 seems to have net sleep-promoting effects, especially with sleep maintenance. The increase in daytime sleep observed in mutant flies may be a compensation mechanism to cope with the reduced amount of nighttime sleep. Further experiments are needed to determine whether the Dα1^KO mutant has intact sleep homeostatic regulation, and whether loss of sleep affects sleep quality. Sleep deprivation has been linked to various detrimental effects, namely reduced life span and learning deficits (Somers, 2017).

Longevity of Dα1^KO mutants was measured, revealing a much shorter life span in the mutant compared to the wild-type. While longevity is not a direct measure of fitness, the mutants' reduced life span highlights the importance of the Dα1 subunit in D. melanogaster physiology. It is not clear if this effect is due to a single physiological role of Dα1, such as the subunits involvement in sleep, or cumulative effects of several impaired physiological roles that impact the mutant's longevity. In either case, it suggests that a resistance allele in the Dα1 subunit is likely to impact the fitness levels of the insect. It also supports the notion that sublethal exposures to insecticides that target receptor subunits orthologous to Dα1 encountered by beneficial insects, such as honey bees, are likely to affect their behavior in ways that may also impact their fitness (Somers, 2017).

The Dα1^KO mutant generated in this study provides a useful tool to study the role of Dα1 in insecticide resistance but, more significantly, to explore the endogenous roles of this gene (Somers, 2017).

Based on prior evidence, it was expected that the Dα1^KO mutant would be resistant to neonicotinoid insecticides (Perry, 2008). However, the data presented in this study allow a fresh evaluation of the value of Dα1 and orthologs in other species as insecticide targets. The ability of the Dα1 knockout flies to survive at all presents a potential issue for resistance evolution to compounds that target this receptor. Given that a wide range of mutations would lead to a total loss-of-function phenotype, such mutations will arise frequently, conferring insecticide resistance. The data presented in this study indicate that, looking beyond viability, there is a significant fitness cost associated with the total loss of Dα1 function, most obvious in terms of severe mating behavior defects, but possibly contributed to by the sleep and longevity phenotypes. Under optimal environmental conditions in the laboratory, large phenotypic differences between Dα1^KO and control flies were observed. It is possible that the fitness cost associated with a null allele of this gene would be even greater under less ideal conditions experienced by natural populations. Therefore, while a nonsense mutation resulting in a resistance allele may be viable, it would likely be associated with fitness costs that would prevent it from persisting in the field. In contrast, mutations that may be null alleles have been found in Dα6 orthologs in spinosad-resistant insects in a number of species. Therefore, it is possible that the spectrum of mutations in Dα1 orthologs that can increase in frequency to confer insecticide resistance in a pest species are constrained by fitness costs (Somers, 2017).

The potential impact of neonicotinoid insecticides on the behavior of beneficial insects, such as A. mellifera, has been intensively researched. Eleven nAChR subunits have been identified in A. mellifera, including a 1:1 ortholog of Dα1. While it cannot be assumed that the functional roles of the honey bee ortholog are identical to those of Dα1, it is likely to influence a range of behaviors that may be perturbed upon exposure to sufficient concentrations of neonicotinoid insecticides (Somers, 2017).

The genetic resources that can be developed to study gene function in D. melanogaster, such as those described here, are extremely powerful. Research on nonmodel insects, both beneficial and pest species, is more challenging. Some functional analysis of Dα6 orthologs from pest species has been possible following the appropriate expression of these genes in a D. melanogaster Dα6 null mutant. A similar approach may be useful in the functional characterization of the orthologs of Dα1 (Somers, 2017).

Insecticides can be powerful probes in neuroscience. To exert toxic effects, these chemicals bind to receptors that have crucial roles in neurotransmission. Research using pyrethroids and cyclodiene insecticides has been productive in elucidating the function of sodium channels and ligand-gated chloride channels, respectively. Similarly, the use of insecticides that target nAChRs has stimulated research on their function. The data demonstrates that such research will make a vital contribution in providing a detailed knowledge of the role neurotransmission in a wide range of behaviors. The variety of behavioral traits impacted by Dα1 loss-of-function indicates a high level of involvement of Dα1 in several behavioral neural circuits, which will require further investigation. Further analysis of Dα1 and the remaining members of the nAChR gene family with a range of paradigms is likely to reveal function in a wide range of insect behaviors (Somers, 2017).

A single photoreceptor splits perception and entrainment by cotransmission

Vision enables both image-forming perception, driven by a contrast-based pathway, and unconscious non-image-forming circadian photoentrainment, driven by an irradiance-based pathway. Although two distinct photoreceptor populations are specialized for each visual task, image-forming photoreceptors can additionally contribute to photoentrainment of the circadian clock in different species. However, it is unknown how the image-forming photoreceptor pathway can functionally implement the segregation of irradiance signals required for circadian photoentrainment from contrast signals required for image perception. This study reports that the Drosophila R8 photoreceptor separates image-forming and irradiance signals by co-transmitting two neurotransmitters, histamine and acetylcholine. This segregation is further established postsynaptically by histamine-receptor-expressing unicolumnar retinotopic neurons and acetylcholine-receptor-expressing multicolumnar integration neurons. The acetylcholine transmission from R8 photoreceptors is sustained by an autocrine negative feedback of the cotransmitted histamine during the light phase of light-dark cycles. At the behavioural level, elimination of histamine and acetylcholine transmission impairs R8-driven motion detection and circadian photoentrainment, respectively. Thus, a single type of photoreceptor can achieve the dichotomy of visual perception and circadian photoentrainment as early as the first visual synapses, revealing a simple yet robust mechanism to segregate and translate distinct sensory features into different animal behaviours (Xiao, 2023).

This study has shown that R8 photoreceptors split visual perception and circadian photoentrainment by co-transmitting two neurotransmitters. This visual segregation is further supported by postsynaptic circuitry in the medulla, such that each unicolumnar neuron mainly receives histaminergic inputs from a single R8 photoreceptor (thus transmitting the fine retinotopic signal), whereas each multicolumnar accessory medulla-innervating, multicolumnar and arcuate neuron (AMA neuron) integrates cholinergic inputs from up to 100 R8 photoreceptor cells (thus integrating the irradiance visual signal). These AMA neurons directly excite downstream clock neurons, forming a shallow three-node circuit for circadian photoentrainment. Thus, this clock-entrainment circuit integrates irradiance signals directly from conventional photoreceptors, bypassing the downstream image-forming processing circuit to avoid a less efficient reconstruction of irradiance signals from the already highly processed signals in the contrast-encoding visual pathways. Notably, the AMA neurons overlap with recently discovered xCEOs that sustain circadian timekeeping of free-running circadian clocks (Tang, 2022). Together, these findings reveal that the AMA neurons and xCEOs play crucial roles in circadian timekeeping, resetting circadian clocks to synchronize their endogenous rhythms to local time by integrating irradiance signals from retinal photoreceptors under LD cycles and sustaining free-running circadian timekeeping through their intrinsic rhythmic electrical oscillations under constant darkness (Xiao, 2023).

The observation that the compound eye-driven electrical responses in clock neurons were reduced by half in the absence of histamine receptors suggests that conventional photoreceptors other than R8 photoreceptors (for example, R1-R6) can use histamine-mediated circuitry pathways to excite clock neurons for circadian photoentrainment. The downstream circuits of R1-R6 photoreceptors might reconstruct irradiance signals from image-forming signals via yet-to-be-uncovered mechanisms as mammalian rod and cone pathways do, indicating the mechanisms underlying irradiance encoding for circadian photoentrainment by conventional photoreceptor pathways might have evolved convergently (Xiao, 2023).

This study also identified an unexpected crosstalk between the image-forming vision and circadian photoentrainment. The chloride channel HisCl1 mediates negative feedback of histamine in R8 photoreceptor cells, thus dynamically reducing photoreceptor depolarization during long light stimulation. This feedback regulation tunes ACh release to avoid its local depletion so that the irradiance signal can be continuously transmitted from R8 photoreceptors to clock neurons during the entire light phase (Xiao, 2023).

This work demonstrates that visual perception and circadian photoentrainment can be segregated as early as the first-order synapses in the visual system, providing a simple yet robust mechanism to enact distinct sensory functions. Furthermore, although cotransmission or co-release of neurotransmitters is an emerging principle in brain research, its behavioural significance remains largely unknown. These finding that cotransmission from the same photoreceptor cells enables segregation and translation of distinct visual features into different behaviours also paves the way for understanding this key, indispensable aspect of the nervous system (Xiao, 2023).

Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila

Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. This study provides the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (gamma-aminobutyric acid) neurons required the acetylcholine receptor nAChRalpha1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, the conserved transcription factor Twist was identified as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, this study has defined how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury (Khuong, 2019).

The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor α1 subunit predicts resistance emergence in pests

Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, this study has investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, the possibility exists of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit (Shimada, 2020)

Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster

Studies about the relationship between substances consumed by humans and their impact on health, in animal models have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolating certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. This work explores the impact of tobacco consumption on a model of "smoking flies". These experiments were designed to provide information about the effects of tobacco consumption on cardiac physiology. Intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation, was assessed. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and alpha 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data would help to understand the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila would help to know the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function (Santilla, 2021).

A single amino acid polymorphism in the Drosophila melanogaster Dalpha1 (ALS) subunit enhances neonicotinoid efficacy at Dalpha1-chicken beta2 hybrid nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. This study investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 mμM ACh, of the Dalpha1-chicken beta2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of Dalpha1 with these neonicotinoids (Ihara, 2014).

Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster

Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. This study focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but this study found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel (Perry, 2008).

Search PubMed for articles about Drosophila Dalpha1

Acebes, A., Grosjean, Y., Everaerts, C. and Ferveur, J. F. (2004). Cholinergic control of synchronized seminal emissions in Drosophila. Curr Biol 14(8): 704-710. PubMed ID: 15084286

Ihara, M., Shimazu, N., Utsunomiya, M., Akamatsu, M., Sattelle, D. B. and Matsuda, K. (2014). A single amino acid polymorphism in the Drosophila melanogaster Dalpha1 (ALS) subunit enhances neonicotinoid efficacy at Dalpha1-chicken beta2 hybrid nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes. Biosci Biotechnol Biochem 78(4): 543-549. PubMed ID: 25036948

Khuong, T. M., Wang, Q. P., Manion, J., Oyston, L. J., Lau, M. T., Towler, H., Lin, Y. Q. and Neely, G. G. (2019). Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila. Sci Adv 5(7): eaaw4099. PubMed ID: 31309148

Perry, T., Heckel, D. G., McKenzie, J. A. and Batterham, P. (2008). Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster. Insect Biochem Mol Biol 38(5): 520-528. PubMed ID: 18405830

Santalla, M., Pagola, L., Gomez, I., Balcazar, D., Valverde, C. A. and Ferrero, P. (2021). Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster. Biol Open. PubMed ID: 33431431

Shimada, S., Kamiya, M., Shigetou, S., Tomiyama, K., Komori, Y., Magara, L., Ihara, M. and Matsuda, K. (2020). The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor α1 subunit predicts resistance emergence in pests. Sci Rep 10(1): 7529. PubMed ID: 32371996

Somers, J., Luong, H. N., Mitchell, J., Batterham, P. and Perry, T. (2017). Pleiotropic effects of loss of the Dα1 subunit in Drosophila melanogaster: Implications for insecticide resistance. Genetics 205(1): 263-271. PubMed ID: 28049707

Tang, M., Cao, L. H., Yang, T., Ma, S. X., Jing, B. Y., Xiao, N., Xu, S., Leng, K. R., Yang, D., Li, M. T., Luo, D. G. (2022). An extra-clock ultradian brain oscillator sustains circadian timekeeping. Sci Adv, 8(35):eabo5506 PubMed ID: 36054358

Xiao, N., Xu, S., Li, Z. K., Tang, M., Mao, R., Yang, T., Ma, S. X., Wang, P. H., Li, M. T., Sunilkumar, A., Rouyer, F., Cao, L. H., Luo, D. G. (2023). A single photoreceptor splits perception and entrainment by cotransmission. Nature, 623(7987):562-570 PubMed ID: 37880372

date revised: 12 December 2024

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