Several Classes Of Insecticides Research Articles

CRISPR/Cas9 mediated validation of spermatogenesis-related gene, tssk2 as a component of genetic pest management of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

Invasive insect pests, currently, pose a serious economic threat to several staple crops all over the world, one such being the fall armyworm, Spodoptera frugiperda. It was first observed in Africa since 2016, outside of its natural habitat in the Americas. Subsequently, it invaded several countries in South and South East Asia and also very recently in Australia. In all the newly invaded regions, maize is the principal crop attacked causing a serious economic concern to the poor farmers, particularly in the developing countries. Owing to the innate genetic ability, it defies many of the management options that include insecticides, Bt transgenics, and so forth. This is due to its high mobility, polyphagy and ability for quick development of resistance to several classes of insecticides. At this critical juncture, CRISPR/Cas9 mediated genome editing has shown a lot of promise in developing a novel area-wide pest management strategy called precision-guided sterile insect technique (pgSIT). pgSIT was initially demonstrated in Drosophila melanogaster which holds a greater promise for the environmentally friendly management of several globally significant agricultural pests such as S. frugiperda. Therefore, before developing both sgRNA and Cas9 transgenic lines, we have validated the target gene such as tssk2 through a non-transgenic approach by microinjecting ribo nucleo protein complex (Cas9 protein and tssk2 sgRNA) into G0 eggs of S. frugiperda. In the current investigation, we have obtained five edited males with distinct mutations which were further used for crossing studies to ascertain the effect of tssk2 editing affecting egg hatchability.

A Review on Fall Armyworm (Spodoptera frugiperda) Insecticide Resistance

The commonly known fall armyworm (Spodoptera frugiperda) is an important pest of corn, cotton, and other crops in the Americas, Africa, and Asia. The species is native to tropical and subtropical regions of the Americas, but it has spread to other parts of the world in recent years. The pest has a wide host range, including important crops like corn, cotton, rice, sorghum, and vegetables. Large populations of fall armyworms can cause significant damage to crops, leading to reduced yields and lower crop quality. The threat of insecticide resistance in S. frugiperda is a growing concern for agricultural production and food security. Insecticides are an important tool for managing fall armyworm populations, but the development of resistance to these chemicals has become a major issue in many regions. This pest has shown resistance to several classes of insecticides, including pyrethroids, organophosphates, and carbamates. To effectively manage insecticide resistance in S. frugiperda populations, it is important to consider these factors and develop integrated pest management strategies that take them into account. This may include the use of alternative control methods, such as biological control and cultural control, and the judicious use of insecticides to minimize selection pressure for resistance development. It is important for farmers, researchers, and policymakers to work together to develop and implement integrated pest management strategies that can effectively manage S. frugiperda populations while minimizing the development and spread of insecticide resistance. Keywords: Fall Armyworm, Spodoptera frugiperda, Insecticide Resistance

Resistance monitoring and mechanism in the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) for chlorantraniliprole from Sichuan Province, China.

The fall armyworm, Spodoptera frugiperda (Noctuidae: Lepidoptera), is a wide-reaching notorious insect pest of important cereal crops, which has developed resistance to multiple classes of insecticides. It invaded the Sichuan Province of China in 2019. In this study, we performed resistance monitoring of insecticides for 11 field-collected populations from Sichuan, and all the populations were susceptible to emamectin benzoate and chlorpyrifos. The variations in resistance level to indoxacarb (resistance ratio (RR), 9.23-45.53-fold), spinetoram (RR, 4.32-18.05-fold), and chlorantraniliprole (RR, 2.02-10.39-fold) were observed among these populations. To investigate the resistance mechanism of chlorantraniliprole, synergism tests were performed and showed that piperonyl butoxide had a slight synergistic effect on chlorantraniliprole for the QJ-20 population (1.43-fold) in moderate resistance (RR, 10.39-fold) compared with the treatment group without synergist. Furthermore, the expression scanning for resistance-related genes showed that five P450 genes (CYP6AE43, CYP321A8, CYP305A1, CYP49A1, and CYP306A1) and the ryanodine receptor gene (Ryr, chlorantraniliprole target) were overexpressed in the QJ-20 population. These results indicated that the fall armyworm in Sichuan has exhibited diverse susceptibilities to several classes of insecticides, and the overexpression of Ryr and several P450 genes may contribute to the development of resistance in S. frugiperda to chlorantraniliprole.

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

Knockdown of a Nicotinic Acetylcholine Receptor Subunit Gene Bdorβ1 Decreases Susceptibility to Oxa-Bridged trans- instead of cis-Nitromethylene Neonicotinoid Insecticides in Bactrocera dorsalis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate the fast action of acetylcholine in synaptic cholinergic transmissions. Insect nAChRs are the target of several classes of insecticides. Here, the full-length cDNA encoding a nAChR beta1 subunit (Bdorβ1) was identified and characterized from a destructive pest, Bactrocera dorsalis. The amino acid sequence of Bdorβ1 shows high identities to other insect nAChRs β1 subunits. Double injection of dsBdorβ1 reduced the expression of Bdorβ1 and in turn significantly decreased susceptibility to oxa-bridged trans- instead of cis-nitromethylene neonicotinoids. Our results support the involvement of Bdorβ1 in the susceptibility of B. dorsalis to oxa-bridged trans- instead of cis-nitromethylene neonicotinoids and imply that these two classes of neonicotinoids might be acting at different nAChR subtypes.

The P450 genes of the cat flea, Ctenocephalides felis: a CYPome in flux.

The genome of the cat flea, an ectoparasite of major veterinary importance and the first representative of the Siphonaptera, is highly unusual among arthropod genomes in showing a variable size and a very large number of gene duplications (Driscoll etal., 2020). The cat flea is the target of several classes of insecticides, justifying the description of its CYPome, the complement of P450s that are an important family of detoxification enzymes. 103 P450 genes were annotated on the nine chromosomes, with an additional 12 genes on small, extrachromosomal scaffolds. Only 34 genes were found as single sequences, with 47 duplicated two to four-fold. This included duplication of genes that are mostly single copy P450 genes in other arthropods. Large clusters of mitochondrial clan P450s were observed, resulting in a CYP12 bloom within this clan to 34 genes, a number of mitochondrial P450s not seen in other animals so far. The variable geometry of the cat flea CYPome poses a challenge to the study of P450 function in this species, and raises the question of the underlying causes of single copy control versus multicopy licence of P450 genes.

Tarnished Plant Bug (Heteroptera: Miridae) Behavioral Responses to Chemical Insecticides

Simple SummaryThe tarnished plant bug, Lygus lineolaris (L.), is a pernicious pest of cotton across the Mid-South and Southeastern US Cotton Belt. In order to manage this pest, numerous insecticide applications are required annually. This has led to widespread resistance and increased cost of control. We tested the behavioral response of tarnished plant bug against various classes of chemistry used for control. The insects avoided certain chemicals and were slightly attracted to others. These findings help us understand the role of tarnished plant bug behavior on field control failures, insecticide resistance, and insecticide resistance management.The tarnished plant bug (Lygus lineolaris Palisot de Beauvois) is the dominant insect pest of cotton (Gossypium hirsutum L.) in the Mid-South Cotton Belt. This is partly due to the fact that this pest has developed resistance to most insecticides used for control. Laboratory experiments were conducted during 2014 and 2015 to study the behavioral response of tarnished plant bug nymphs to several classes of insecticides. Twenty third-instar nymphs were placed in individual dishes divided into four quadrants with five green bean pieces in each quadrant (10 treated and 10 untreated green beans in each dish). Dishes were checked at 1, 4, 8, and 24 h. Tarnished plant bug nymphs appeared to avoid green beans treated with IGR, pyrethroid, organophosphate, or carbamate insecticides, while there appeared to be an attraction to green bean pieces treated with sulfoxamine and pyridine carboxamide insecticides. No relationship was observed with neonicotinoid insecticides within 24 h.

Evaluation of 21 Thailand Maize Germplasms for Resistance to Leaf Feeding Spodoptera frugiperda (Lepidoptera: Noctuidae)

Native to the Americas, the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera:Noctuidae), is a recent invasive pest of Africa and Asia. This insect causes economic damage to maize (Zea mays L.) primarily through leaf and ear tissue feeding. The pest is resistant to several classes of insecticide and several Bt-maize toxins. Native resistance in maize to fall armyworm could be used with existing control tactics to enhance control of this insect. The objective for this study was to evaluate maize germplasm, developed from Thailand and maintained by the U.S. National Plant Germplasm System, for resistance to leaf feeding fall armyworm. Plants were grown in the field and artificially infested at the seven-leaf stage with fall armyworms. Visual rating scores for leaf feeding damage were recorded at 7 and 14 days post infestation. Scores for maize germplasm Ames 26254 were not different than the resistant check, GT-FAWCC(C5), and germplasm PI 506347 and PI 690324 were not significantly different from Ames 26254. In total, five Thailand maize germplasms received 14-day damage scores of ≤ 6.0 across both years of testing and were considered moderately resistant to fall armyworm leaf feeding. These maize germplasms are tropical and require breeding to adapt progeny for research in temperate areas.

Three sodium channel mutations from Aedes albopictus confer resistance to Type I, but not Type II pyrethroids

Voltage-gated sodium channels are the major targets of several classes of insecticides, including pyrethroids. However, sensitivities of many insect pest species to pyrethroids have gradually decreased due to overuse in pest management programs. One major mechanism of pyrethroid resistance known as knockdown resistance (kdr) involves mutations in the sodium channel gene. Three new mutations in helix IIIS6 of sodium channel (I1532T and F1534S/L) are recently detected in several pyrethroid-resistant populations of Aedes albopictus. The roles of these mutations in pyrethroid resistance have not been functionally examined. We introduced mutations I1532T and F1534S/L alone or in combination into the pyrethroid-sensitive sodium channel AaNav1-1 from Aedes aegypti by site-directed mutagenesis and explored effects of these mutations on the channel gating and sensitivity to pyrethroids. No significant modifications in channel properties were detected, except for a slightly changed activation by F1534S and I1532T + F1534S. However, I1532T and F1534S/L substantially reduced the channel sensitivity to Type I pyrethroids, permethrin and bifenthrin, but not to two Type II pyrethroids, deltamethrin and cypermethrin. The double mutations did not increase the channel resistance to permethrin or bifenthrin. We have built a Nav1.4-based homology model of the AaNav1-1 channel and docked pyrethroids in the model to explain different sensitivities of the mutants to Type I and Type II pyrethroids. The results will assist in developing molecular markers for monitoring pest resistance to pyrethroids. They also provide new insight in the molecular basis of different action of Type I and Type II pyrethroids on sodium channels.

Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia

BackgroundThe movement of malaria vectors into new areas is a growing concern in the efforts to control malaria. The recent report of Anopheles stephensi in eastern Ethiopia has raised the necessity to understand the insecticide resistance status of the vector in the region to better inform vector-based interventions. The aim of this study was to evaluate insecticide resistance in An. stephensi in eastern Ethiopia using two approaches: (1) World Health Organization (WHO) bioassay tests in An. stephensi; and (2) genetic analysis of insecticide resistance genes in An. stephensi in eastern Ethiopia.MethodsMosquito larvae and pupae were collected from Kebri Dehar. Insecticide susceptibility of An. stephensi was tested with malathion 5%, bendiocarb 0.1%, propoxur 0.1%, deltamethrin 0.05%, permethrin 0.75%, pirimiphos-methyl 0.25% and DDT 4%, according to WHO standard protocols. In this study, the knockdown resistance locus (kdr) in the voltage gated sodium channel (vgsc) and ace1R locus in the acetylcholinesterase gene (ace-1) were analysed in An. stephensi.ResultsAll An. stephensi samples were resistant to carbamates, with mortality rates of 23% and 21% for bendiocarb and propoxur, respectively. Adult An. stephensi was also resistant to pyrethroid insecticides with mortality rates 67% for deltamethrin and 53% for permethrin. Resistance to DDT and malathion was detected in An. stephensi with mortality rates of 32% as well as An. stephensi was resistance to pirimiphos-methyl with mortality rates 14%. Analysis of the insecticide resistance loci revealed the absence of kdr L1014F and L1014S mutations and the ace1R G119S mutation.ConclusionOverall, these findings support that An. stephensi is resistant to several classes of insecticides, most notably pyrethroids. However, the absence of the kdr L1014 gene may suggest non-target site resistance mechanisms. Continuous insecticide resistance monitoring should be carried out in the region to confirm the documented resistance and exploring mechanisms conferring resistance in An. stephensi in Ethiopia.

Evaluation of XL370A-Derived Maize Germplasm for Resistance to Leaf Feeding by Fall Armyworm1

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is an economically important insect with larvae damaging maize (Zea mays L.) leaves and ear tissue. The pest has become resistant to several classes of insecticide and Bt-maize grown in some geographical areas. Once discovered and characterized, native sources of maize resistance to this pest could be effectively integrated with existing control tactics. The objective for this study was to test experimental lines derived from maize germplasm XL370A for resistance to leaf feeding by fall armyworm. Plants were grown in the field in 2018 and 2019, artificially infested with fall armyworm, and leaf damage scores recorded. Average 14-day scores for experimental maize lines GEMN-0095 (5.8), GEMN-0096 (5.7), and GEMN-0133 (5.6) were moderately resistant and 7- and 14-day scores for these entries were not significantly different across both years. Cuba 94 was not significantly different from the three entries with the exception of having greater 7-day damage scores in 2019. GEMN-0048 was not resistant but variability was observed in 14-day scores between 4 (resistant) and 8 (susceptible) in individual plants. The experimental lines are adapted for growth in temperate regions and might provide maize breeding programs with useful levels of resistance to fall armyworm.

Distinct functional properties of sodium channel variants are associated with usage of alternative exons in Nilaparvata lugens

Voltage-gated sodium channels (Nav) are essential for electrical signaling in the nervous system. They are also the primary targets of several classes of insecticides including pyrethroids. There is only one sodium channel gene in most insect species, whereas mammals possess at least nine sodium channel genes. Extensive alternative splicing and RNA editing of sodium channel transcripts have been documented in many insect species. However, the functional consequences of these post-transcriptional events have been evaluated only in DmNav and BgNav from Drosophila melanogaster and Blattella germanica, respectively. In this study, we isolated 41 full-length cDNA clones encoding 34 sodium channel (NlNav) variants from a major rice pest, the brown planthopper (Nilaparvata lugens Stål). The 34 NlNav variants represent 24 distinct splicing types based on the usage of nine alternative exons, six of which, including exon b, have been previously reported in other insect species. When expressed in Xenopus oocytes, NlNav variants lacking exon b generated significantly larger sodium currents than variants possessing exon b, suggesting an inhibitory effect of exon b on sodium current expression. A similar effect has been reported for exon b from BgNav. Mutational analysis showed that three conserved amino acid residues encoded by exon b are critical for its inhibitory effect. In addition, mutually exclusive exons k/l contribute to distinct functional properties and channel sensitivity to pyrethroids. Altogether, these results show that alternative splicing generates functional diversity of sodium channels in this insect species and that the role of exon b in regulating neuronal excitability is likely conserved among insect species.

Insecticidal effect of aconitine on the rice brown planthoppers.

The brown planthopper, Nilaparvata lugens (Stål), severely damages rice production and develops high level resistance to several classes of insecticides. To find potential insecticidal resources is always important. As an environmentally friendly compound, aconitine exhibits potential pesticide features. In the present study, the pesticide and knockdown effects of aconitine were first tested on the brown planthopper. The results showed that the knockdown rates for an aconitine concentration of 200 ppm was 83.6%. The insecticidal LD50 was 22.68 ng/pest (95% CI, 17.75–28.99). The molecular mechanisms responding to aconitine application were analyzed through transcriptional sequencing. Compared to that of the knockdown nymphs of the brown planthoppers, the enzymes CYP3A4, UDP-glucuronosyltransferase (UGT), GST, carboxylesterase (EC3.1.1.1), and GABAergic synapse were up-regulated. We inferred that aconitine might be neurotoxic to the brown planthoppers, and the conscious nymphs resist the drug neurotoxicity through the upregulation of CYP3A4, UGT, and GABA receptor mutation. Although aconitine is not safe for mammals, it may be a leading compound to develop novel insecticides.

Evaluation of Maize Germplasm from Saint Croix for Resistance to Leaf Feeding by Fall Armyworm1

Maize (Zea mays L.) is a preferred host of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera:Noctuidae), with larvae primarily feeding on developing leaves and ear tissue. The fall armyworm is resistant to several classes of insecticide and Bt-maize grown in certain areas. Native sources of plant resistance to the pest are available for public use, but new sources of resistance need to be discovered and developed. The objective for this study was to test maize germplasm collected from Saint Croix, U.S. Virgin Islands, for resistance to leaf feeding by fall armyworm. Plants were grown in the field and artificially infested at a high level. Scores of damage by fall armyworm feeding on leaves at 7 and 14 days differed significantly for the 13 maize genotypes tested. Scores at 14 days for Saint Croix Group 1 (5.8), Saint Croix Group 3 (5.6), Saint Croix 2 (5.6), and Saint Croix 7 (6.0) were moderately resistant and not significantly different from one another. Individual plants in the populations were variable for resistance to leaf feeding, and scored between 4 and 7. It should be possible to select within the populations for greater resistance to damage by fall armyworms feeding on leaves.

Cloning and Functional Characterisation of the Duplicated RDL Subunits from the Pea Aphid, Acyrthosiphon pisum.

The insect GABA receptor, RDL (resistance to dieldrin), is a cys-loop ligand-gated ion channel (cysLGIC) that plays a central role in neuronal signaling, and is the target of several classes of insecticides. Many insects studied to date possess one Rdl gene; however, there is evidence of two Rdls in aphids. To characterise further this insecticide target from pests that cause millions of dollars’ worth of crop damage each year, we identified the complete cysLGIC gene superfamily of the pea aphid, Acyrthosiphon pisum, using BLAST analysis. This confirmed the presence of two Rdl-like genes (RDL1 and RDL2) that likely arose from a recent gene duplication. When expressed individually in Xenopus laevis oocytes, both subunits formed functional ion channels gated by GABA. Alternative splicing of RDL1 influenced the potency of GABA, and the potency of fipronil was different on the RDL1bd splice variant and RDL2. Imidacloprid and clothianidin showed no antagonistic activity on RDL1, whilst 100 μM thiacloprid reduced the GABA responses of RDL1 and RDL2 to 55% and 62%, respectively. It was concluded that gene duplication of Rdl may have conferred increased tolerance to natural insecticides, and played a role in the evolution of insect cysLGICs.

Mechanisms of Action, Resistance and Toxicity of Insecticides Targeting GABA Receptors.

γ-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor. We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included. The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue.

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b5 enhanced the kcat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min−1) with little effect on KM (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.

Partitioning the roles of CYP6G1 and gut microbes in the metabolism of the insecticide imidacloprid in Drosophila melanogaster

Resistance to insecticides through enhanced metabolism is a worldwide problem. The Cyp6g1 gene of the vinegar fly, Drosophila melanogaster, is a paradigm for the study of metabolic resistance. Constitutive overexpression of this gene confers resistance to several classes of insecticides, including the neonicotinoid imidacloprid (IMI). The metabolism of IMI in this species has been previously shown to yield oxidative and nitro-reduced metabolites. While levels of the oxidative metabolites are correlated with CYP6G1 expression, nitro-reduced metabolites are not, raising the question of how these metabolites are produced. Some IMI metabolites are known to be toxic, making their fate within the insect a second question of interest. These questions have been addressed by coupling the genetic tools of gene overexpression and CRISPR gene knock-out with the mass spectrometric technique, the Twin-Ion Method (TIM). Analysing axenic larvae indicated that microbes living within D. melanogaster are largely responsible for the production of the nitro-reduced metabolites. Knock-out of Cyp6g1 revealed functional redundancy, with some metabolites produced by CYP6G1 still detected. IMI metabolism was shown to produce toxic products that are not further metabolized but readily excreted, even when produced in the Central Nervous System (CNS), highlighting the significance of transport and excretion in metabolic resistance.

Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance.

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

Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies

Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.