Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Swift regulation of nicotinic acetylcholine receptors (nAChRs) and glutathione S-transferase (GST) enables the rapid detoxification of thiacloprid in pine sawyer beetles.

Thiacloprid, a neonicotinoid insecticide, has become one of the major control agents for the pine sawyer beetle, Monochamus alternatus Hope, however, the mechanism of detoxification is unknown. We demonstrate that glutathione S-transferases (GSTs) and nicotinic acetylcholine receptors (nAChRs) are involved in the rapid detoxification of thiacloprid in M. alternatus larvae. The activity of detoxification enzyme GSTs was significantly higher, while the activity of acetylcholinesterase (AChE) was inhibited under thiacloprid exposure. The inhibition of AChE activity led to lethal over-stimulation of the cholinergic synapse, which was then released by the rapid downregulation of nAChRs. Meanwhile, GSTs were overexpressed to detoxify thiacloprid accordingly. A total of 3 nAChR and 12 GST genes were identified from M. alternatus, among which ManAChRα2 and MaGSTs1 were predicted to confer thiacloprid tolerance. RNA interference (RNAi) was subsequently conducted to confirm the function of ManAChRα2 and MaGSTs1 genes in thiacloprid detoxification. The successful knock-down of the ManAChRα2 gene led to lower mortality of M. alternatus under LC30 thiacloprid treatment, and the suppression of the MaGSTs1 gene increased the mortality rate of M. alternatus. However, the mortality rate has no significant difference with controls when thiacloprid was fed together with both dsMaGSTs1 and dsManAChRα2. Molecular docking modeled the molecular basis for interaction between MaGSTs1/ManAChR and thiacloprid. This study highlights the important roles that ManAChRα2 and MaGSTs1 genes play in thiacloprid detoxification through transcriptional regulation and enzymatic metabolization, and proposes a new avenue for integrated pest management that combines pesticides and RNAi technology as an efficient strategy for M. alternatus control.

Signaling through the nicotinic acetylcholine receptor in the liver protects against the development of metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of liver steatosis, the most common liver disease, and substantially increases the mortality rate. However, limited therapies are currently available to prevent MASH development. Identifying potential pharmacological treatments for the condition has been hampered by its heterogeneous and complex nature. Here, we identified a hepatic nonneuronal cholinergic signaling pathway required for metabolic adaptation to caloric overload. We found that cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) is highly expressed in hepatocytes of mice and humans. Further, CHRNA2 is activated by a subpopulation of local acetylcholine-producing macrophages during MASH development. The activation of CHRNA2 coordinates defensive programs against a broad spectrum of MASH-related pathogenesis, including steatosis, inflammation, and fibrosis. Hepatocyte-specific loss of CHRNA2 signaling accelerates the disease onset in different MASH mouse models. Activation of this pathway via pharmacological inhibition of acetylcholine degradation protects against MASH development. Our study uncovers a hepatic nicotinic cholinergic receptor pathway that constitutes a cell-autonomous self-defense route against prolonged metabolic stress and holds therapeutic potential for combatting human MASH.

Unravelling nicotinic receptor and ligand features underlying neonicotinoid knockdown actions on the malaria vector mosquito Anopheles gambiae.

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.

Identifying and assessing a prognostic model based on disulfidptosis-related genes: implications for immune microenvironment and tumor biology in lung adenocarcinoma.

Introduction: Lung cancer, with the highest global mortality rate among cancers, presents a grim prognosis, often diagnosed at an advanced stage in nearly 70% of cases. Recent research has unveiled a novel mechanism of cell death termed disulfidptosis, which is facilitated by glucose scarcity and the protein SLC7A11. Methods: Utilizing the least absolute shrinkage and selection operator (LASSO) regression analysis combined with Cox regression analysis, we constructed a prognostic model focusing on disulfidptosis-related genes. Nomograms, correlation analyses, and enrichment analyses were employed to assess the significance of this model. Among the genes incorporated into the model, CHRNA5 was selected for further investigation regarding its role in LUAD cells. Biological functions of CHRNA5 were assessed using EdU, transwell, and CCK-8 assays. Results: The efficacy of the model was validated through internal testing and an external validation set, with further evaluation of its robustness and clinical applicability using a nomogram. Subsequent correlation analyses revealed associations between the risk score and infiltration of various cancer types, as well as oncogene expression. Enrichment analysis also identified associations between the risk score and pivotal biological processes and KEGG pathways. Our findings underscore the significant impact of CHRNA5 on LUAD cell proliferation, migration, and disulfidptosis. Conclusion: This study successfully developed and validated a robust prognostic model centered on disulfidptosis-related genes, providing a foundation for predicting prognosis in LUAD patients.

Bulk segregant mapping and transcriptome analyses reveal the molecular mechanisms of spinetoram resistance in Spodoptera frugiperda.

The evolution of resistance to insecticides poses a significant threat to pest management programs. Understanding the molecular mechanisms underlying insecticide resistance is essential to design sustainable pest control and resistance management programs. The fall armyworm, Spodoptera frugiperda, is an important insect pest of many crops and has a remarkable ability to evolve resistance to insecticides. In this study, we employed bulk segregant analysis (BSA) combined with DNA and RNA sequencing to characterize the molecular basis of spinetoram resistance in S. frugiperda. Analysis of genomic data derived from spinetoram selected and unselected bulks and the spinetoram-resistant and susceptible parental strains led to the identification of a three-nucleotide deletion in the gene encoding the nicotinic acetylcholine receptor α6 subunit (nAChR α6). Transcriptome profiling identified the upregulation of few genes encoding detoxification enzymes associated with spinetoram resistance. Thus, spinetoram resistance in S. frugiperda appears to be mediated mainly by target site insensitivity with a minor role of detoxification enzymes. Our findings provide insight into the mechanisms underpinning resistance to spinetoram in S. frugiperda and will inform the development of strategies to control this highly damaging, globally distributed crop pest.

Investigating CHRNA5, CHRNA3, and CHRNB4 variants in the genetic landscape of substance use disorder in Jordan.

BACKGROUND: Substance use disorder (SUD) is a complex illness that can be attributed to the interaction between environmental and genetic factors. The nicotinic receptor gene cluster on chromosome 15 has a plausible association with SUD, particularly with nicotine dependence. METHODS: This study investigated 15 SNPs within the CHRNA5, CHRNA3, and CHRNB4 genes. Sequencing was used for genotyping 495 Jordanian males with SUD and 497 controls matched for age, gender, and descent. RESULTS: Our findings revealed that none of the tested alleles or genotypes were correlated with SUD. However, our analysis suggests that the route of substance use was linked to rs1051730 (P value = 0.04), rs8040868 (P value = 0.01) of CHRNA3, and rs16969968 (P value = 0.03) of CHRNA5. Additionally, a correlation was identified between rs3813567 of the CHRNB4 gene and the age at substance use onset (P value = 0.04). CONCLUSIONS: Variants in CHRNA5, CHRNA3, and CHRNB4 may interact with SUD features that can influence the development and progression of the disorder among Jordanians.

Abundant extrasynaptic expression of α3ß4-containing nicotinic acetylcholine receptors in the medial habenula-interpeduncular nucleus pathway in mice.

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and ß4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3ß4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and ß4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and ß4 subunits disappeared in α5ß4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that α3ß4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3ß4-containing nAChRs in the crucial pathway of nicotine dependence.

Spermatogonial stem cells in the 129 inbred strain exhibit unique requirements for self-renewal.

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Aspartic acid mutagenesis of αO-Conotoxin GeXIVA isomers reveals arginine residues crucial for inhibition of the α9α10 nicotinic acetylcholine receptor.

The two most active disulfide bond isomers of the analgesic αO-conotoxin GeXIVA, namely GeXIVA[1, 2] and GeXIVA[1, 4], were subjected to Asp-scanning mutagenesis to determine the key amino acid residues for activity at the rat α9α10 nicotinic acetylcholine receptor (nAChR). These studies revealed the key role of arginine residues for the activity of GeXIVA isomers towards the α9α10 nAChR. Based on these results, additional analogues with 2-4 mutations were designed and tested. The analogues [T1A,D14A,V28K]GeXIVA[1, 2] and [D14A,I23A,V28K]GeXIVA[1, 4] were developed and showed sub-nanomolar activity for the α9α10 nAChR with IC50 values of 0.79 and 0.38 nM. The latter analogue had exceptional selectivity for the α9α10 receptor subtype over other nAChR subtypes and can be considered as a drug candidate for further development. Molecular dynamics of receptor-ligand complexes allowed us to make deductions about the possible causes of increases in the affinity of key GeXIVA[1, 4] mutants for the α9α10 nAChR.

Alpha9alpha10 knockout mice show altered physiological and behavioral responses to signals in masking noise.

Medial olivocochlear (MOC) efferents modulate outer hair cell motility through specialized nicotinic acetylcholine receptors to support encoding of signals in noise. Transgenic mice lacking the alpha9 subunits of these receptors (α9KOs) have normal hearing in quiet and noise, but lack classic cochlear suppression effects and show abnormal temporal, spectral, and spatial processing. Mice deficient for both the alpha9 and alpha10 receptor subunits (α9α10KOs) may exhibit more severe MOC-related phenotypes. Like α9KOs, α9α10KOs have normal auditory brainstem response (ABR) thresholds and weak MOC reflexes. Here, we further characterized auditory function in α9α10KO mice. Wild-type (WT) and α9α10KO mice had similar ABR thresholds and acoustic startle response amplitudes in quiet and noise, and similar frequency and intensity difference sensitivity. α9α10KO mice had larger ABR Wave I amplitudes than WTs in quiet and noise. Other ABR metrics of hearing-in-noise function yielded conflicting findings regarding α9α10KO susceptibility to masking effects. α9α10KO mice also had larger startle amplitudes in tone backgrounds than WTs. Overall, α9α10KO mice had grossly normal auditory function in quiet and noise, although their larger ABR amplitudes and hyperreactive startles suggest some auditory processing abnormalities. These findings contribute to the growing literature showing mixed effects of MOC dysfunction on hearing.

α5-nAChR/ADAM10 signaling mediates nicotine-related cutaneous melanoma progression via STAT3 activation.

Skin cutaneous melanoma (SKCM) is the skin malignancy with the highest mortality rate, and its morbidity rate is on the rise worldwide. Smoking is an independent marker of poor prognosis in melanoma. The α5-nicotinic acetylcholine receptor (α5-nAChR), one of the receptors for nicotine, is involved in the proliferation, migration and invasion of SKCM cells. Nicotine has been reported to promote the expression of a disintegrin and metalloproteinase 10 (ADAM10), which is the key gene involved in melanoma progression. Here, we explored the link between α5-nAChR and ADAM10 in nicotine-associated cutaneous melanoma. α5-nAChR expression was correlated with ADAM10 expression and lower survival in SKCM. α5-nAChR mediated nicotine-induced ADAM10 expression via STAT3. The α5-nAChR/ADAM10 signaling axis was involved in the stemness and migration of SKCM cells. Furthermore, α5-nAChR expression was associated with ADAM10 expression, EMT marker expression and stemness marker expression in nicotine-related mice homograft tissues. These results suggest the role of the α5-nAChR/ADAM10 signaling pathway in nicotine-induced melanoma progression.

Functions and pharmacology of α2ß2 nicotinic acetylcholine receptors; in and out of the shadow of α4ß2 nicotinic acetylcholine receptors.

Although α2 was the first neuronal nicotinic acetylcholine receptor (nAChR) receptor subunit to be cloned, due to its low level of expression in rodent brain, its study has largely been neglected. This study provides a comparison of the α2 and α4 structures and their functional similarities, especially in regard to the existence of low and high sensitivity forms based on subunit stoichiometry. We show that the pharmacological profiles of the low and high sensitivity forms of α2ß2 and α4ß2 receptors are very similar in their responses to nicotine, with high sensitivity receptors showing protracted responses. Sazetidine A, an agonist that is selective for the high sensitivity α4 receptors also selectively activates high sensitivity α2 receptors. Likewise, α2 receptors have similar responses as α4 receptors to the positive allosteric modulators (PAMs) desformylflustrabromine (dFBr) and NS9283. We show that the partial agonists for α4ß2 receptors, cytisine and varenicline are also partial agonists for α2ß2 receptors. Studies have shown that levels of α2 expression may be much higher in the brains of primates than those of rodents, suggesting a potential importance for human therapeutics. High-affinity nAChR have been studied in humans with PET ligands such as flubatine. We show that flubatine has similar activity with α2ß2 and α4ß2 receptors so that α2 receptors will also be detected in PET studies that have previously presumed to selectively detect α4ß2 receptors. Therefore, α2 receptors need more consideration in the development of therapeutics to manage nicotine addiction and declining cholinergic function in age and disease.

Computational Modeling Oriented Substructure Splicing Application in the Identification of Thiazolidine Derivatives as Potential Low Honeybee Toxic Neonicotinoids.

With the aim of identifying novel neonicotinoid insecticides with low bee toxicity, a series of compounds bearing thiazolidine moiety, which has been shown to be low bee toxic, were rationally designed through substructure splicing strategy and evaluated insecticidal activities. The optimal compounds A24 and A29 exhibited LC50 values of 30.01 and 17.08 mg/L against Aphis craccivora, respectively. Electrophysiological studies performed on Xenopus oocytes indicated that compound A29 acted on insect nAChR, with EC50 value of 50.11 µM. Docking binding mode analysis demonstrated that A29 bound to Lymnaea stagnalis acetylcholine binding protein through H-bonds with the residues of D_Arg55, D_Leu102, and D_Val114. Quantum mechanics calculation showed that A29 had a higher highest occupied molecular orbit (HOMO) energy and lower vertical ionization potential (IP) value compared to the high bee toxic imidacloprid, showing potentially low bee toxicity. Bee toxicity predictive model also indicated that A29 was nontoxic to honeybees. Our present work identified an innovative insecticidal scaffold and might facilitate the further exploration of low bee toxic neonicotinoid insecticides.

Potency and Target Surface Interaction of Diazinoyl Nicotinic Insecticides.

Structure-activity relationships of diazinoyl nicotinic insecticides (diazinoyl isomers and 5- or 6-substituted pyrazin-2-oyl analogues) are considered in terms of affinity to the insect nicotinic acetylcholine receptor (nAChR) and insecticidal activity against the imidacloprid-resistant brown planthopper. Among the test compounds, 3-(6-chloropyridin-3-ylmethyl)-2-(pyrazinoyl)iminothiazoline shows the highest potency in nAChR affinity and insecticidal activity. Aplysia californica acetylcholine binding protein (AChBP) mutants (Y55W + Q57R and Y55W + Q57T) are utilized to compare molecular recognition of nicotinic insecticides with diverse pharmacophores. N-nitro- or N-cyanoimine imidacloprid or acetamiprid, respectively, exhibits a high affinity to these AChBP mutants at a similar potency level. Intriguingly, the pyrazin-2-oyl analogue has a higher affinity to AChBP Y55W + Q57R than that to Y55W + Q57T, thereby indicating that pyrazine nitrogen atoms contact Arg57 guanidinium and Trp55 indole NH. Furthermore, nicotine prefers AChBP Y55W + Q57T over Y55W + Q57R, conceivably suggesting that the protonated nicotine is repulsed by Arg57 guanidinium, consistent with its inferior potency to insect nAChR.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and ß1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

Pathogenic residue insertion in neuronal nicotinic receptor alters intra- and inter-subunit interactions that tune channel gating.

We describe molecular-level functional changes in the α4ß2 nicotinic acetylcholine receptor by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single-channel currents reveal the primary effect is to stabilize the open channel state, while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4)2(ß2)3 and (α4)3(ß2)2, indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intrasubunit and intersubunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together, the results reveal a novel structural region of the α4ß2 nicotinic acetylcholine receptor in which interhelical interactions tune the stability of the open channel state.

Survey of target site mutations linked with insecticide resistance in Italian populations of Aphis gossypii.

BACKGROUND: Aphis gossypii is a worldwide agricultural pest that causes high levels of economic losses by feeding and transmitting virus diseases. It is usually controlled by chemical insecticides, but this could lead to the selection of resistant populations. Several single nucleotide polymorphisms (SNPs) have been identified associated with insecticide resistance. Monitoring activities to detect the presence of such mutations in field populations can have an important role in insect pest management but, currently, no information on Italian strains is available. RESULTS: The presence of target site mutations conferring resistance to different insecticides was analysed in Italian field collected populations of A. gossypii with an allele specific approach (QSGG, Qualitative Sybr-Green Genotyping). Primers were designed to detect mutations in genes coding acetylcholinesterase (S431F), nicotinic acetylcholine receptor (R81T) and voltage-gated sodium channel (M918L and L1014F). S431F was widespread but with high variability across populations. R81T was detected for the first time in Italy but only in two populations. The L1014F mutation (kdr) was not found, while in the samples showing the M918L two different nucleotidic substitutions were detected. Mutant allele frequencies were, respectively, 0.70 (S431), 0.31 (M918) and 0.02 (R81). Further analysis on the voltage-gated sodium channel gene showed the presence of eight haplotypes and one non-synonymous mutation in the gene coding region. CONCLUSION: Multiple target-site mutations were detected within Italian populations. The combinations of genotypes observed in certain locations could affect negatively the control of this pest. Preliminary insights on the genetic structure in the Italian populations of A. gossypii were acquired. © 2024 Society of Chemical Industry.