A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis

High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad‐resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance‐associated mutation (G275E) is predicted to lie at the top of the third α‐helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate‐gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance‐associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action.     

Mutation in the ace‐1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a major invasive pest that has spread throughout many countries in the Mediterranean basin and parts of Asia over the last decade. The control of T. absoluta has relied heavily on the use of chemical insecticides, a strategy that has led to the evolution of resistance. In this study, biological and molecular methods were used to determine the susceptibility of five strains of T. absoluta to the organophosphate chlorpyrifos and to investigate the molecular mechanisms underlying resistance to this class of insecticides. High levels of resistance to chlorpyrifos were observed in all five strains tested. Cloning and sequencing of the gene encoding the organophosphate target site, ace‐1, of T. absoluta revealed the presence of an alanine to serine substitution at a position that has been previously linked with organophosphate resistance across a range of different insect and mite species. The presence of this mutation at high frequency in T. absoluta populations originating from various countries further supports the suggestion that the rapid expansion of this species is, in part, mediated by the resistance of this pest to chemical insecticides.     

Rapsyn‐mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high‐density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the α subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn‐induced clustering of the AChR β, γ, and δ subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into αδ dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild‐type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn‐mediated clustering of an assembly intermediate, the αδ dimer. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 486–501, 2003     

Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.     

Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.     

Essential Oils as Biocides for the Control of Fungal Infections and Devastating Pest (Tuta absoluta) of Tomato (Lycopersicon esculentum Mill.)

Thymus capitatus and Tetraclinis articulata essential oils as well their major components (carvacrol and α‐pinene) were evaluated for their antifungal and insecticidal activities. Both oils showed good in vitro antifungal activity against Fusarium oxysporum, Aspergillus niger, Penicillium sp., Alternaria alternata, and Botrytis cinerea, the fungi causing tomato rot. In vivo results indicate the efficacies of both essential oils and carvacrol of reduce postharvest fungal pathogens, such as B. cinerea and Al. alternata that are responsible of black and gray rot of tomato fruit. Disease incidence of Al. alternata and B. cinerea decreased on average from 55% to 80% with essential oil of Th. capitatus and pure carcvacrol, while Te. articulata essential oil exhibited inhibition of fungal growth of 55% and 25% against Al. alternata and B. cinerea, respectively, with concentration of 0.4 μl/l air. The insecticidal activity of Th. capitatus and Te. articulata essential oils exhibited also a good insecticidal activity. At the concentration of 0.2 μl/ml air, the oils caused mortality over 80% for all larval stages of Tuta absoluta and 100% mortality for the first‐instar after 1.5 h only of exposure. α‐Pinene presented lower insecticidal and antifungal activities compared to essential oils of Th. capitatus, Te. articulata and pure carvacrol. Thus, these essential oils can be used as a potential source to develop control agents to manage some of the main pests and fungal diseases of tomato crops.     

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [³H]-phencyclidine ([³H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [³H]-ACh or ¹²⁵I-α-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [³H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh or carbamylcholine-stimulated [³H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [³H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.     

Sugars as complementary alternative food for the establishment of Nesidiocoris tenuis in greenhouse tomato

Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is an omnivorous generalist predator which is augmentatively released and conserved for control of whiteflies (Hemiptera: Aleyrodidae) and Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in tomato crops. Eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) are often provided as factitious prey to improve the establishment of N. tenuis after its release. We first tested different amounts of E. kuehniella eggs per plant to optimize N. tenuis establishment and then investigated whether the amount of eggs that optimized N. tenuis establishment might be reduced by adding sugars (hydrocapsules filled with 0.5 m sucrose) under walk‐in cage and commercial greenhouse conditions. These experiments demonstrated that the addition of sugar to the diet of N. tenuis could half the amount of E. kuehniella eggs required to establish N. tenuis. Under greenhouse conditions, the progeny of N. tenuis per plant did not differ significantly between E. kuehniella alone or the half amount of E. kuehniella plus hydrocapsules. These results demonstrated that the sugar could partially substitute for E. kuehniella eggs improve establishment of N. tenuis and suggest that natural sugars such as nectar and honeydew might also beneficial.     

A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F

As well as generating protein isoform diversity, in some cases alternative splicing generates RNAs that harbor premature termination codons and that are subject to nonsense-mediated decay (NMD). We previously identified an apparent pseudo-exon in the rat α-tropomyosin (Tpm1) gene as a probable genuine alternatively spliced exon that causes NMD when spliced into Tpm1 RNA. Here, we report the analysis of cis-acting splicing regulatory elements within this “nonsense exon.” Guided by the data set of predicted splicing enhancer and silencer elements compiled by Zhang and Chasin, we made a series of mutations through the nonsense exon and found that like authentic exons it is densely packed with enhancer and silencer elements. Strikingly, 11 of 13 tested mutations behaved as predicted computationally. In particular, we found that a G-rich silencer at the 5′ end, which is crucial for skipping of the nonsense exon, functions by binding hnRNP-H and F.     

Population growth potential of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) on tomato,potato, and eggplant

The influence of host plant on population dynamics of an invasive pest, Tuta absoluta was studied on three economically important solanaceous crops. Experiments were conducted in laboratory (29 ± 0.5°C, 75 ± 5% RH and a photoperiod of 14:10 hr [L:D]) using tomato (Solanum lycopersicum L.), potato (Solanum tuberosum L.) and eggplant (Solanum melongena L.). Results indicated that intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R₀) were higher, and mean generation time (T) was the shortest on tomato. Results suggested that T. absoluta developed on all the three plants, and tomato plant was most preferred one. Results suggested that T. absoluta has a potential to become a serious pest on potato and even on eggplant under favourable conditions. We used the life tables of 0.025th and 0.975th percentiles of bootstraps to project the uncertainty of population growth, a new concept.     

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are members of the Cys‐loop ligand‐gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α‐helical segments (M1–M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X‐ray structure of the first eukaryotic Cys‐loop receptor, a truncated (intracellular domain missing) glutamate‐gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel‐lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2–M3 alignments as observed in X‐ray structures of prokaryotic Gloeobacter violaceus ligand‐gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys‐loop receptors is conserved between prokaryotes and eukaryotes.     

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR‐NLR immune receptor

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Ry_sto, from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Ry_sto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Ry_sto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single‐molecule real‐time sequencing). Ry_sto was found to encode a nucleotide‐binding leucine‐rich repeat (NLR) protein with an N‐terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Ry_sto‐dependent extreme resistance was temperature‐independent and requires EDS1 and NRG1 proteins. Ry_sto may prove valuable for creating PVY‐resistant cultivars of potato and other Solanaceae crops.     

Novel alternative splicing of GABA receptor RDL exon 9 from Laodelphax striatellus modulates agonist potency

The resistance to dieldrin gene (RDL) encodes the primary subunit of the insect ionotropic γ-aminobutyric acid (GABA) receptor (GABAR), which is the target of phenylpyrazole and isoxazoline insecticides. The splice variants in exons 3 and 6 of RDL, which have been widely explored in many insects, modulate the agonist potency of the homomeric RDL GABAR and potentially play an important role in the development of insects. In the present study, four splice variants of exon 9 were identified in RDL of the small brown planthopper, Laodelphax striatellus (LsRDL), resulting in LsRDL-9a, LsRDL-9a’, LsRDL-9b, and LsRDL-9c. LsRDL-9a has one more amino acid (E, glutamic acid) compared with LsRDL-9a’, and LsRDL-9b lacked two amino acids and had seven different amino acids compared with LsRDL-9c. Two-electrode voltage-clamp recording on LsRDLs expressed in Xenopus oocytes showed that alternative splicing of exon 9 has significant impact on LsRDL sensitivity to the agonists GABA and β-alanine, whereas no significant difference was observed in the potencies of the non-competitive antagonists (NCAs) ethiprole and fluralaner on the splice variants. Our results suggest that alternative splicing of RDL exon 9 broadens functional capabilities of the GABAR in L. striatellus by influencing the action of GABA.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

Insulin resistance induces earlier initiation of cognitive dysfunction mediated by cholinergic deregulation in a mouse model of Alzheimer's disease

Although insulin resistance increases the risk of Alzheimer's disease (AD), the mechanisms remain unclear, partly because no animal model exhibits the insulin-resistant phenotype without persistent hyperglycemia. Here we established an AD model with whole-body insulin resistance without persistent hyperglycemia (APP/IR-dKI mice) by crossbreeding constitutive knock-in mice with P1195L-mutated insulin receptor (IR-KI mice) and those with mutated amyloid precursor protein (App^NL-G-F mice: APP-KI mice). APP/IR-dKI mice exhibited cognitive impairment at an earlier age than APP-KI mice. Since cholinergic dysfunction is a major characteristic of AD, pharmacological interventions on the cholinergic system were performed to investigate the mechanism. Antagonism to a nicotinic acetylcholine receptor α7 (nAChRα7) suppressed cognitive function and cortical blood flow (CBF) response to cholinergic-regulated peripheral stimulation in APP-KI mice but not APP/IR-dKI mice. Cortical expression of Chrna7, encoding nAChRα7, was downregulated in APP/IR-dKI mice compared with APP-KI. Amyloid β burden did not differ between APP-KI and APP/IR-dKI mice. Therefore, insulin resistance, not persistent hyperglycemia, induces the earlier onset of cognitive dysfunction and CBF deregulation mediated by nAChRα7 downregulation. Our mouse model will help clarify the association between type 2 diabetes mellitus and AD.     

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3' splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3' splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3' splice sites.     

Organophosphate and pyrethroid resistances in the tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) from Iran

The tomato leafminer, Tuta absoluta (Meyrich) (Lepidoptera: Gelechiidae), is a serious pest of tomato crops worldwide. The intensive use of chemical pesticides to control it has led to the selection of resistant populations. This study investigated the resistance of T. absoluta populations to pyrethroid and the organophosphate insecticides from ten regions of Iran. The resistance ratios at LC₅₀ for chlorpyrifos and diazinon varied among populations from 4.3 to 12 and from 1.4 to 9.0, respectively. The resistance ratios of the pyrethroids cypermethrin, deltamethrin and permethrin varied from 1.3 to 3.7, 2.7 to 13 and 1.2 to 4.3, respectively. Inclusion of synergists in toxicological bioassays and the variation observed in the activity of esterases, glutathione S‐transferase and cytochrome P450‐dependent monooxygenase suggest the existence of metabolically based resistance. Esterase and P450 biochemical assays were positively correlated with deltamethrin, and cypermethrin tolerance and diazinon tolerance correlated with esterase activity. The genes encoding the organophosphate and pyrethroid target sites acetylcholinesterase (ace1) and sodium channel (kdr) were partly sequenced. The genotyping revealed mutations in high frequencies in all populations leading to an A201S substitution in ace1 and three substitutions in the sodium channel gene L1014F, M918T, T929I. In summary, our results indicate the presence of organophosphate and pyrethroid resistance in Iranian T. absoluta populations with involvement of both detoxification enzymes and target site alterations. Most likely the populations of T. absoluta imported to Iran were resistant upon arrival.     

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.