Insect Central Nervous System Research Articles

Toxicity evaluation of neonicotinoids to earthworm (Eisenia fetida) behaviors by a novel locomotion tracking assay

Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed Eisenia fetida to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94–68.63% and increment in freezing time movement ratio by 1.51–4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24–70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94–12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.

Molecular insights into the interaction between myoglobin and Imidacloprid: Multi-spectral experiments and computational simulations

Imidacloprid (IMI) belongs to the neonicotinoid insecticides, specifically the imidazole chemical group, which exhibits a high level of contact, digestive, and systemic effects on the central nervous system of insects. In this study, we investigated the impact of IMI on the conformation and structural stability of myoglobin (Mb) protein using spectroscopic techniques combined with molecular docking and simulation approaches. Additionally, we examined the thermal stability of the resulting complex. The UV–Visible results demonstrated the interaction between IMI and Mb protein. The intrinsic fluorescence of Mb was effectively quenched by IMI, indicating successful binding and the formation of a stable complex. Thermodynamic analysis revealed that the spontaneous bonding process was primarily governed by van der Waals forces and hydrogen bonds. FT-IR analysis indicated that IMI induced conformational changes in Mb, providing further evidence of its potential toxicity and structural damage to Mb. Furthermore, the interaction significantly enhanced the thermal stability of Mb compared to Mb alone. Molecular docking revealed the preferred binding site of IMI within the Mb structure. Molecular dynamics (MD) simulation results further supported the findings by demonstrating a decrease in compactness and an increase in the stability of the Mb-IMI complex. These findings offer valuable insights into the potential toxicity risks of IMI to human health and shed light on the binding mechanism between IMI and Mb. The results of this study serve as a useful model for gaining a deeper understanding of the effects of these pesticides on proteins and body health.

Critical roles of nicotinic acetylcholine receptors in olfactory memory formation and retrieval in crickets.

Acetylcholine (ACh) is a major excitatory neurotransmitter in the insect central nervous system, and insect neurons express several types of ACh receptors (AChRs). AChRs are classified into two subgroups, muscarinic AChRs and nicotinic AChRs (nAChRs). nAChRs are also divided into two subgroups by sensitivity to α-bungarotoxin (α-BGT). The cricket Gryllus bimaculatus is one of the useful insects for studying the molecular mechanisms in olfactory learning and memory. However, the roles of nAChRs in olfactory learning and memory of the cricket are still unknown. In the present study, to investigate whether nAChRs are involved in cricket olfactory learning and memory, we tested the effects of two different AChR antagonists on long-term memory (LTM) formation and retrieval in a behavioral assay. The two AChR antagonists that we used are mecamylamine (MEC), an α-BGT-insensitive nAChR antagonist, and methyllycaconitine (MLA), an α-BGT-sensitive nAChR antagonist. In crickets, multiple-trial olfactory conditioning induced 1-day memory (LTM), whereas single-trial olfactory conditioning induced 1-h memory (mid-term memory, MTM) but not 1-day memory. Crickets injected with MEC 20min before the retention test at 1day after the multiple-trial conditioning exhibited no memory retrieval. This indicates that α-BGT-insensitive nAChRs participate in memory retrieval. In addition, crickets injected with MLA before the multiple-trial conditioning exhibited MTM but not LTM, indicating that α-BGT-sensitive nAChRs participate in the formation of LTM. Moreover, injection of nicotine (an nAChR agonist) before the single-trial conditioning induced LTM. Finally, the nitric oxide (NO)-cGMP signaling pathway is known to participate in the formation of LTM in crickets, and we conducted co-injection experiments with an agonist or inhibitor of the nAChR and an activator or inhibitor of the NO-cGMP signaling pathway. The results suggest that nAChR works upstream of the NO-cGMP signaling system in the LTM formation process.

Whole-Mount Immunofluorescent Labeling of the Mosquito Central Nervous System.

Mosquito-borne disease is a major global public health issue. One path toward the development of evidence-based strategies to limit mosquito biting is the study of the mosquito nervous system-in particular, the sensory systems that drive biting behavior. The central nervous system of insects consists of the brain and the ventral nerve cord. Here, we describe a protocol for dissecting, immunofluorescent labeling, and imaging both of these structures in the mosquito. This protocol was optimized for Aedes aegypti and works well on Anopheles gambiae tissue. It has not been tested in other mosquito species, but we anticipate that it would work on a range of mosquitoes, and, if not, our protocol will provide a starting point from which to optimize. Notably, a limited number of antibodies cross-react with Ae. aegypti proteins. This protocol is intended for use with validated antibodies and can also be used to test new antibodies as they are generated. It has been successfully used to visualize protein tags, such as green fluorescent protein, that have been introduced into the mosquito to amplify or detect their presence.

Shisa reduces the sensitivity of homomeric RDL channel to GABA in the two-spotted spider mite, Tetranychus urticae Koch

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and TuShisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated TuRDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC50 = 53.34 μM). No GABA-activated current could be observed in TuShisa-expressed oocytes, whereas TuShisa could reduce the sensitivity of TuRDL/TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of TuRDL and TuShisa were built by the SWISS-MODEL server, their interaction was predicted using Z-DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of TuShisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the TuShisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa.

Functional interactions between potassium-chloride cotransporter (KCC) and inward rectifier potassium (Kir) channels in the insect central nervous system

The K+/Cl− cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular chloride (Cl−) concentration and has been shown to be functionally coupled to the GABA-gated chloride channels (GGCC) in Drosophila central neurons. Further, pharmacological inhibition of KCC has been shown to lead to acute toxicity of mosquitoes that highlights the toxicological relevance of insect KCC. Yet, gaps in knowledge remain regarding physiological drivers of KCC function and interactions of ion flux mechanisms upstream of GGCC in insects. Considering this, we employed electrophysiological and fluorescent microscopy techniques to further characterize KCC in the insect nervous system. Fluorescent microscopy indicated insect KCC2 is expressed in rdl neurons, which is the neuron type responsible for GABA-mediated neurotransmission, and are coexpressed with inward rectifier potassium (Kir) 2 channels. Coexpression of Kir2 and KCC2 suggested the possibility of functional coupling between these two K+ flux pathways. Indeed, extracellular recordings of Drosophila CNS showed pre-block of Kir channels prior to block of KCC led to a significant (P < 0.001) increase in CNS firing rates over baseline that when taken together, supports functional coupling of Kir to KCC function. Additionally, we documented a synergistic increase to toxicity of VU0463271, an established KCC inhibitor, above the expected additive toxicity after co-treatment with the Kir inhibitor, VU041. These data expand current knowledge regarding the physiological roles of KCC and Kir channels in the insect nervous system by defining additional pathways that facilitate inhibitory neurotransmission through GGCC.

Embryonic toxicity of Imidacloprid: Impact on hatchability, survivability, swimming speed and cardiac function of catfish, Clarias gariepinus

Imidacloprid is a systemic insecticide that belongs to the neonicotinoid class of chemicals that act on the central nervous system of insects. Imidacloprid is used to control sucking insects, chewing insects such as termites, soil insects, and fleas on pets, as well as to treat structures, crops, soil, and seeds. As a result of these factors, this pesticide may end up in the aquatic environment via municipal discharges and runoff. Although the presence of imidacloprid in aquatic environments has been underreported as widespread, the toxic effects of this pesticide may have serious implications on aquatic organisms, particularly at environmentally relevant concentrations and demand more attention. Given this knowledge, the goal of this study was to investigate the effects of imidacloprid on Clarias gariepinus embryonic development. Clarias gariepinus embryos (3 h post-fertilization) were exposed to environmentally relevant concentrations of imidacloprid (10, 30, 100, and 500 µg/L) until 48 h post-fertilization using a modified fish embryo acute toxicity test (OECD TG 236). A stereomicroscope was used to assess hatchability, deformity, heart rate, and swimming speed as endpoints. According to our results of the developmental acute toxicity test, imidacloprid significantly reduced the hatching rate and heartbeats of C. gariepinus embryos. It also influenced the swimming kinematics of exposed embryos and caused teratogenic effects such as yolk sac rupture, pericardial oedema, lordosis, an abnormally shaped head, and altered epiboly. Our results allow us to conclude that imidacloprid is a toxic pesticide in the early life stages of C. gariepinus due to its high teratogenic potential.

Giant Cortical Glial Cells in the Central Nervous System of Insects

Giant Cortical Glial Cells in the Central Nervous System of Insects

The modulatory effects of biogenic amines on male mating performance in Bactrocera dorsalis.

In insects, the emergence of mating behavior requires the interplay among sex-determination hierarchy mechanisms that regulate sex-specific differentiation, perception and integration of different sensory cues, and precisely patterned behavioral outputs. Biogenic amines, including octopamine (OA), dopamine (DA), tyramine (TA), serotonin and histamine, have been identified and proposed as putative neurotransmitters, neurohormones and/or neuromodulators in the central nervous system of insects to influence multiple physiologies and behaviors. The current study provides the physiological roles and pharmacology of these biogenic amines in the mating performance of Bactrocera dorsalis. Silencing gene expressions coding for biosynthetic enzymes of DA and serotonin in male flies could decrease mating rates, while OA, TA and histamine had no such effects on mating. Furthermore, injection of DA or the DA receptor antagonist chlorpromazine could affect mating rate, as well as injection of serotonin. Pharmacological treatments with other biogenic amines or their receptor antagonists in male flies have no roles in regulating mating performance. We conclude that DA and its receptors are involved in regulating male mating behaviors in B. dorsalis, while changes in serotonin levels in male flies could also affect mating rates. In the current study, the modulatory effects of these biogenic amines on mating performance were investigated, and these results will be helpful in providing a new strategy for controlling B. dorsalis.

Metabolic detoxification and ace-1 target site mutations associated with acetamiprid resistance in Aedes aegypti L.

Despite the continuous use of chemical interventions, Aedes-borne diseases remain on the rise. Neonicotinoids are new, safer, and relatively effective pharmacological interventions against mosquitoes. Neonicotinoids interact with the postsynaptic nicotinic acetylcholine receptors (nAChRs) of the insect central nervous system, but the absence of nAChR polymorphism in resistant phenotypes makes their involvement in neonicotinoid resistance uncertain. Thus, an investigation was carried out to understand the role of metabolic detoxification and target site insensitivity in imparting acetamiprid resistance in Aedes aegypti larvae. Studies were conducted on the parent susceptible strain (PS), acetamiprid-larval selected strain for five generations (ACSF-5; 8.83-fold resistance) and 10 generations (ACSF-10; 19.74-fold resistance) of Ae. aegypti. The larval selection raised α-esterase and β-esterase activities by 1.32-fold and 1.34-fold, respectively, in ACSF-10 as compared to PS, while the corresponding glutathione-S-transferase and acetylcholinesterase activity increased by 22.5 and 2%. The ace-1 gene in PS and ACSF-10 showed four mismatches in the 1312—1511 bp region due to mutations in the Y455C codon (tyrosine to cysteine) at the 1367th position (TAC→TGC); I457V codon (isoleucine to valine) at 1372 bp and 1374 bp (ATA→GTG); and R494M codon (arginine to methionine) at 1484 bp (AGG→ATG). The R494M mutation was the novel and dominant type, observed in 70% ACSF-10 population, and has not been reported so far. The studies evidenced the combination of metabolic detoxification and target site mutation in imparting acetamiprid resistance in Ae. aegypti.

The Golgi Method.

Silver staining by the Golgi method is a classical procedure to identify the three-dimensional morphology of individual neurons. Although the method was developed in the 1870s, it is still used to study the morphological characteristics of neuron types in the central nervous system, either alone or in combination with other neuroanatomical techniques. The neuropil of insects is fully accessible to the original refractive staining procedure, and modifications of the Golgi technique have paved the path for modern structural research on the insect central nervous system. Here, we provide an introduction to this easy and low-cost method.

Neurogenesis in the Insect Central Nervous System and Its Peculiarities in the Brain Mushroom Bodies

Neurogenesis in the Insect Central Nervous System and Its Peculiarities in the Brain Mushroom Bodies

Seasonal variations of serotonin in the visual system of an ant revealed by immunofluorescence and a machine learning approach.

Hibernation, as an adaptation to seasonal environmental changes in temperate or boreal regions, has profound effects on mammalian brains. Social insects of temperate regions hibernate as well, but despite abundant knowledge on structural and functional plasticity in insect brains, the question of how seasonal activity variations affect insect central nervous systems has not yet been thoroughly addressed. Here, we studied potential variations of serotonin-immunoreactivity in visual information processing centres in the brain of the long-lived ant species Lasius niger. Quantitative immunofluorescence analysis revealed stronger serotonergic signals in the lamina and medulla of the optic lobes of wild or active laboratory workers than in hibernating animals. Instead of statistical inference by testing, differentiability of seasonal serotonin-immunoreactivity was confirmed by a machine learning analysis using convolutional artificial neuronal networks (ANNs) with the digital immunofluorescence images as input information. Machine learning models revealed additional differences in the third visual processing centre, the lobula. We further investigated these results by gradient-weighted class activation mapping. We conclude that seasonal activity variations are represented in the ant brain, and that machine learning by ANNs can contribute to the discovery of such variations.

Nicotinic acetylcholine receptor modulator insecticides act on diverse receptor subtypes with distinct subunit compositions.

Insect nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of many insecticides, including neonicotinoids, which are the most widely used insecticides in the world. However, the development of resistance in pests and the negative impacts on bee pollinators affect the application of insecticides and have created a demand for alternatives. Thus, it is very important to understand the mode of action of these insecticides, which is not fully understood at the molecular level. In this study, we systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunit mutants to eleven insecticides acting on nAChRs. Our results showed that there are several subtypes of nAChRs with distinct subunit compositions that are responsible for the toxicity of different insecticides. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. Moreover, spinosyns may act exclusively on the α6 homomeric pentamers but not any other nAChRs. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for the management of resistance and the development of novel insecticides targeting these important ion channels.

IVAN OLEKSIYOVYCH LEVCHENKO. MAN AND HER AFFAIRS

Introduction. One of the ways in which bee colonies interact is through stereotypical dances. Many authors have studied the relationship between dance. However, there was no consensus on their functional significance. Finding out the question of the signal significance of bee dances became the main direction of Ivan Levchenko’s scientific research. In addition to theoretical significance, such studies are needed to develop new methods for controlling the flight-collecting activity of bees. The goal of the work. To trace the main stages of IO Levchenko's life and creative path. To give the main achievements of his research, to characterize their theoretical and practical significance for beekeeping. Materials and methods of research. The basis of this study is the scientific literature on the topic of work, scientific works of domestic and foreign scientists. In the process of research, historical-comparative, analytical-synthetic, as well as the method of source analysis are used. Results of research and discussion. Ivan Levchenko was born on August 31, 1930 in the village of Samara, Azov district, Rostov region, in a large family of workers. The formation of Ivan Lavchenko as a researcher took place at the Kyiv State University named after T.G. Shevchenko (1951‒1956). After graduating from university he worked in the laboratory of arachnoentomology, in the Kaniv Biological Reserve. This was the period of further development of the researcher. During this time, techniques have been developed that make it possible to record the reaction of bees to various processes occurring in the central nervous system of insects. A fruitful period in the scientific activity of Ivan Levchenko were the years of work at the Institute of Zoology named after I. Schmalhausen National Academy of Sciences (1962-1994). Here, in 1966, he defended his dissertation on "The main forms of relationship in bees - collectors." Considering bees' dances as a special form of reflecting the excitation of the bee's nervous system, Ivan Levchenko naturally raised and successfully solved the question of the impact on bee signaling of such factors as air temperature, features of the terrain, bee strength, sugar concentration in nectar, carbohydrate characteristics feed composition. Ivan Levchenko considered the discovery of individual smell in worker bees to be an important factor in his scientific work. Since 1994, I. Levchenko worked at the NSC "Institute of Beekeeping named after P.I. Prokopovich "in the established laboratory of bee ethology. The direction of research has changed, but has always been associated with practical beekeeping. І. Levchenko conducted active training of junior specialists in the field of beekeeping. Under his supervision, six PhD theses were defended. Published 300 scientific papers. Conclusions and prospects for further research. Ivan Levchenko proved by his research that the transmission of information about the coordinates of the source of the bribe in the family of the honey bee is carried out by many factors - smell, acoustic and tactile stimuli, including dancing. An attempt to explain the process of mobilizing bees to collect food with just one stimulus is invalid. We can talk about the relative importance of a factor in a particular situation, but the effect of mobilization in general must be considered as an adequate response of the family to a set of stimuli. Honey bees have undergone a long process of evolution from a solitary form to a highly organized and family with a social - collective way of life. Ways of relations in them include both ancient forms (sight and smell) and new, highly specialized moving reactions - dances of scouts. Accordingly, the search for honey flow by bees can be carried out both at the level of individuals and the family as a whole. But there is no doubt that all forms of search are of great biological importance to the family and together provide rapid mobilization for food collection.

Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.

Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.

Evaluation of Developmental and Reproductive Fitness of Laboratory Selected Thiamethoxam Resistance in Black Legume Aphid Aphis craccivora Koch.

Thiamethoxam is a neonicotinoid group of insecticide which are selective agonists of the insect nicotinic acetylcholine receptor (nAChR), a pentameric cys-loop ligand-gated ion channel located in the central nervous system of insects. They provide farmers with invaluable, highly effective tools against some of the world’s most destructive crop pests. Black legume aphids, Aphis craccivora is one of the devastating polyphagous pests in agroecosystem and to manage their population, farmers are still relying on application of synthetic insecticides like Thiamethoxam. To evaluate the risk of resistance development, selection of A. craccivora was done to characterize Thiamethoxam resistance along with evaluation of developmental and reproductive fitness cost of the resistance. About 86.19 folds of resistance were developed after selecting for 24 generations. In the first twelve generations steady development of resistance was noticed followed by development become stiffer till 16th generation and more or less stable till 24th generations. Relative fitness of the selected resistant strain of aphids was decreased with longer nymphal duration and adult longevity. The mean nymphal duration was increased from 4.35±1.02 days (in F0) to 7.9±0.57 days (in F24). The fecundity rate was significantly less in resistant population (16.0±5.34) as compared to the susceptible strain (54.71±7.63) whereas, oviposition periods were significantly longer (12.49±1.44 days) in resistant population as compare to susceptible strain (9.50±1.22 days).

Self-Poisoning With Safer Insecticide: A Case Series on Imidacloprid Poisoning.

Imidacloprid (IMI) is a systemic insecticide that belongs to the neonicotinoid group of insecticides. It is highly effective against sucking, boring, and root-feeding insects. Besides, it has a favorable toxicological profile on humans. Currently, IMI is the largest selling insecticide in the world by replacing organophosphates and carbamates. It acts as nicotinic acetylcholine receptor agonist in the central nervous system of insects. To date, there is no specific antidote available for IMI poisoning. Despite a better safety profile on humans, severe toxicity from IMI poisoning is not uncommon. Here, we report a series of 4 cases who were admitted to our tertiary care center with a history of IMI poisoning.

Temporal regulation of nicotinic acetylcholine receptor subunits supports central cholinergic synapse development in Drosophila

The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 (Dα1) and nAchRα6 (Dα6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses.

Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.