A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

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.

Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster.

Although the Cas9 protein from Streptococcus pyogenes (SpCas9) is the most widely used clustered regularly interspaced short palindromic repeats (CRISPR) variant in genome engineering experiments, it does have certain limitations. First, the stringent requirement for the protospacer adjacent motif (PAM) sequence limits the target DNA that can be manipulated using this method in insects. Second, its complementarity specifications are not very stringent, meaning that it can sometimes cause off-target effects at the target site. A recent study reported that an evolved SpCas9 variant, xCas9(3.7), with preference for various 5'-NG-3' PAM sequences not only has the broadest PAM compatibility but also has much greater DNA specificity and lower genome-wide off-target activity than SpCas9 in mammalian cells. Here we applied the CRISPR/xCas9 system to target the white gene in Drosophila melanogaster, testing the genome-editing efficiency of xCas9 at different PAM sites. On the GGG PAM site, xCas9 showed less activity than SpCas9. For the non-NGG PAM site TGA, xCas9 could produce DNA cleavage and indel-mediated disruption on the target gene. However, for other non-NGG PAM sites, xCas9 showed no activity. These findings show that the evolved Cas9 variant with broad PAM compatibility is functional in Drosophila to induce heritable gene alterations, increasing the targeting range for the applications of genome editing in insects.

Functional validation of A2'N mutation of the RDL GABA receptor against fipronil via molecular modeling and genome engineering in drosophila.

BACKGROUND: Insect RDL (resistant to dieldrin) receptors are essential pentameric ligand-gated chloride channels that mediate the neuroinhibitory effect of GABA, the chief inhibitory neurotransmitter in the central nervous system. These receptors serve as primary targets for various insecticides, including noncompetitive antagonists (NCAs) such as cyclodiene organochlorines and phenylpyrazoles, as well as allosteric modulators like meta-diamides and isoxazolines. This study focuses on a newly discovered A2'N mutation within the RDL receptors, identified in fipronil-resistant planthoppers. Despite in vitro electrophysiological studies have proposed its role in conferring target-site resistance, in vivo genetic functional validation of this mutation remains unexplored. RESULTS: Our research employed toxicity bioassays, assessing various Rdl genotypes against a spectrum of insecticides, including fipronil, α-endosulfan, broflanilide, and isocycloseram. Results revealed distinct resistance profiles for A2'N and A2'S mutants, indicating different binding interactions of RDL receptors with NCAs. Significantly, the A2'N heterozygote showed substantial resistance to fipronil, despite its homozygous lethality. Molecular modeling and docking simulations further supported these findings, highlighting unique binding poses for fipronil and α-endosulfan. CONCLUSION: This study confirmed that A2'N mutation of the RDL GABA receptor confer high resistance to fipronil in vivo. The observed resistance in A2'N mutants is likely attributable to a steric hindrance mechanism, wherein the introduction of larger side chains hampers fipronil binding, even in a heterozygous state. © 2023 Society of Chemical Industry.

Effects of RDL GABA Receptor Point Mutants on Susceptibility to Meta-Diamide and Isoxazoline Insecticides in Drosophila melanogaster.

Ionotropic γ-aminobutyric acid (GABA) receptors in insects, specifically those composed of the RDL (resistant to dieldrin) subunit, serve as important targets for commonly used synthetic insecticides. These insecticides belong to various chemical classes, such as phenylpyrazoles, cyclodienes, meta-diamides, and isoxazolines, with the latter two potentially binding to the transmembrane inter-subunit pocket. However, the specific amino acid residues that contribute to the high sensitivity of insect RDL receptors to these novel insecticides remain elusive. In this study, we investigated the susceptibility of seven distinct Drosophila melanogaster Rdl point mutants against four meta-diamide and isoxazoline insecticides: isocycloseram, fluxametamide, fluralaner, and broflanilide. Our findings indicate that, despite exhibiting increased sensitivity to fluralaner in vitro, the RdlI276C mutant showed resistance to isocycloseram and fluxametamide. Similarly, the double-points mutant RdlI276F+G279S also showed decreased sensitivity to the tested isoxazolines. On the other hand, the RdlG335M mutant displayed high levels of resistance to all tested insecticides. Molecular modeling and docking simulations further supported these findings, highlighting similar binding poses for these insecticides. In summary, our research provides robust in vivo evidence supporting the idea that the inter-subunit amino acids within transmembrane M1 and M3 domains form the binding site crucial for meta-diamide and isoxazoline insecticide interactions. This study highlights the complex interplay between mutations and insecticide susceptibility, paving the way for more targeted pest control strategies.

Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution.

The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. Here we show that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. We used in vivo genome editing in Drosophila melanogaster to evaluate the fitness effects of each putative resistance mutation and found that pleiotropy both facilitates and constrains the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.

An insecticide target in mechanoreceptor neurons.

Hundreds of neurotoxic insecticides are currently in use. However, only a few direct targets have been identified. Here, using Drosophila and the insecticide flonicamid, we identified nicotinamidase (Naam) as a previous unidentified molecular target for an insecticide. Naam is expressed in chordotonal stretch-receptor neurons, and inhibition of Naam by a metabolite of flonicamid, TFNA-AM (4-trifluoromethylnicotinamide), induces accumulation of substrate nicotinamide and greatly inhibits negative geotaxis. Engineered flies harboring a point mutation in the active site show insecticide resistance and defects in gravity sensing. Bees are resistant to flonicamid because of a gene duplication, resulting in the generation of a TFNA-AM-insensitive Naam. Our results, in combination with the absence of genes encoding Naam in vertebrate genomes, suggest that TFNA-AM and potential species-specific Naam inhibitors could be developed as novel insecticides, anthelmintics, and antimicrobials for agriculture and human health.

An octopamine receptor confers selective toxicity of amitraz on honeybees and Varroa mites.

The Varroa destructor mite is a devastating parasite of Apis mellifera honeybees. They can cause colonies to collapse by spreading viruses and feeding on the fat reserves of adults and larvae. Amitraz is used to control mites due to its low toxicity to bees; however, the mechanism of bee resistance to amitraz remains unknown. In this study, we found that amitraz and its major metabolite potently activated all four mite octopamine receptors. Behavioral assays using Drosophila null mutants of octopamine receptors identified one receptor subtype Octß2R as the sole target of amitraz in vivo. We found that thermogenetic activation of octß2R-expressing neurons mimics amitraz poisoning symptoms in target pests. We next confirmed that the mite Octß2R was more sensitive to amitraz and its metabolite than the bee Octß2R in pharmacological assays and transgenic flies. Furthermore, replacement of three bee-specific residues with the counterparts in the mite receptor increased amitraz sensitivity of the bee Octß2R, indicating that the relative insensitivity of their receptor is the major mechanism for honeybees to resist amitraz. The present findings have important implications for resistance management and the design of safer insecticides that selectively target pests while maintaining low toxicity to non-target pollinators.

Identification, Characterization and Expression Analysis of TRP Channel Genes in the Vegetable Pest, Pieris rapae.

Transient receptor potential (TRP) channels are critical for insects to detect environmental stimuli and regulate homeostasis. Moreover, this superfamily has become potential molecular targets for insecticides or repellents. Pieris rapae is one of the most common and widely spread pests of Brassicaceae plants. Therefore, it is necessary to study TRP channels (TRPs) in P. rapae. In this study, we identified 14 TRPs in P. rapae, including two Water witch (Wtrw) genes. By contrast, only one Wtrw gene exists in Drosophila and functions in hygrosensation. We also found splice isoforms of Pyrexia (Pyx), TRPgamma (TRPγ) and TRP-Melastatin (TRPM). These three genes are related to temperature and gravity sensation, fine motor control, homeostasis regulation of Mg2+ and Zn2+ in Drosophila, respectively. Evolutionary analysis showed that the TRPs of P. rapae were well clustered into their own subfamilies. Real-time quantitative PCR (qPCR) showed that PrTRPs were widely distributed in the external sensory organs, including antennae, mouthparts, legs, wings and in the internal physiological organs, including brains, fat bodies, guts, Malpighian tubules, ovaries, as well as testis. Our study established a solid foundation for functional studies of TRP channels in P. rapae, and would be benefit to developing new approaches to control P. rapae targeting these important ion channels.

Molecular cloning and characterization of TRPVs in two rice pests: Nilaparvata lugens (Stål) and Nephotettix cincticeps (Uhler).

BACKGROUD: Insect TRPV is a subfamily of transient receptor potential (TRP) ion channels, including two genes, nanchung and inactive. It has recently been found that two commercial insecticides, pymetrozine and pyrifluquinazon, target the heteromeric TRPV ion channel complex which is specifically expressed in the chordotonal organ neurons in Drosophila. However, information on the TRPV genes in agricultural insects is still limited. RESULTS: In this study, we cloned and characterized two TRPV genes from Nilaparvata lugens (NlNan and NLIav) and Nephotettix cincticeps (NcNan and NcIav), two serious rice pests throughout Asia. The deduced amino acid sequences share highly identity with other insect homologues (58-85%) and have the characteristic TRPV domain architecture: five ankyrin repeats and six transmembrane domains. These TRPV transcripts were expressed in all developmental stages and expression levels in male adults were significantly higher than in female adults. Moreover, expression levels in antennae were much higher than in heads and legs. CONCLUSION: NlNan, NlIav, NcNan and NcIav may have roles in male-specific behaviors, and the sequence information lays the foundation for further study on the structural and functional characterization of TRPVs in agricultural pests. © 2018 Society of Chemical Industry.

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion.

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered ß1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A 1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.

Synthesis and structure-activity relationship analysis of bicyclophosphorothionate blockers with selectivity for housefly gamma-aminobutyric acid receptor channels.

BACKGROUND: Bicyclophosphorothionates (2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-sulfides) are blockers (or non-competitive antagonists) of gamma-aminobutyric acid (GABA) receptor channels. Twenty-two bicyclophosphorothionates with different 3- and 4-substituents were synthesised, and [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays were performed to evaluate their affinities for housefly and rat GABA receptors. RESULTS: Introduction of an isopropyl group at the 3-position enhanced the affinity of bicyclophosphorothionates for housefly GABA receptors and reduced the affinity towards rat GABA receptors. The 4-isopentyl-3-isopropylbicyclophosphorothionate showed the highest affinity for housefly GABA receptors (IC(50) = 103 nM) among the analogues tested, while the 4-cyclohexylbicyclophosphorothionate showed the highest affinity for rat GABA receptors (IC(50) = 125 nM). Among the bicyclophosphorothionates synthesised to date, the former analogue exhibited the highest selectivity for housefly GABA receptors, with an IC(50)(rat)/IC(50)(fly) ratio of approximately 97. Three-dimensional GABA receptor models successfully explained the structure-activity relationships of the bicyclophosphorothionates. CONCLUSION: The results indicate that minor structural modifications of blockers can change their selectivity for insect versus mammalian GABA receptors. The substituent at the 3-position of the bicyclophosphorothionates dictates selectivity for housefly versus rat GABA receptors. This information should prove useful for the design of safer insecticides and parasiticides.