Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Cis- and trans-acting variants contribute to survivorship in a naïve Drosophila melanogaster population exposed to ryanoid insecticides.

Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster Here we use a naïve population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naïve to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals.

Processing Apples to Puree or Juice Speeds Gastric Emptying and Reduces Postprandial Intestinal Volumes and Satiety in Healthy Adults.

BACKGROUND: Whole apples produce greater satiety than processed apples, but the underlying mechanisms remain unclear. OBJECTIVE: Our aim was to assess the intragastric processing of apple preparations and the associated small and large bowel contents using MRI. METHODS: An open label, 3-way crossover, randomized, controlled trial. Eighteen healthy adults (mean ± SD age, 25 ± 4 y; BMI, 22.7 ± 3.5 kg/m2) underwent serial MRI scans on 3 occasions separated by 7 d, after consumption of isocaloric (178 kcal) portions of either whole apples, apple puree, or apple juice. Gastric emptying, small bowel water content (SBWC; primary endpoint), were measured at baseline and at 45 min intervals (0-270 min) postmeal ingestion. Fullness and satiety were also assessed at each time point. Treatment effects between groups were analyzed using ANOVA. RESULTS: Gastric emptying half-time (GE t50) was greater (P < 0.0001) after participants consumed whole apple (mean ± SEM), 65 (3.3) min compared with when they consumed apple puree (41 [2.8] min) or apple juice (38 [2.9] min), times that did not differ. Postprandial area under the curve (AUC) (135-270 min) SBWC was also greater for whole apples than puree (P = 0.025) and juice (P = 0.0004) but juice and puree did not differ. AUC for fullness and satiety (0-270 min) postingestion was also greater (P = 0.002 and 0.004, respectively) for whole apple compared with juice but juice and puree did not differ. CONCLUSIONS: Gastric emptying is slower after whole apple consumption causing a greater sensation of fullness and satiety than puree or juice in healthy adults. Whole apples increased small bowel and colonic contents during the later phase of the study which may be relevant for subsequent food consumption.This study was registered at clinicaltrials.gov as NCT03714464.

Sublethal larval exposure to imidacloprid impacts adult behaviour in Drosophila melanogaster.

Pesticides are now chronically found in numerous ecosystems incurring widespread toxic effects on multiple organisms. For insects, the larvae are very exposed to pesticide pollution and the acute effect of insecticides on larvae has been characterized in a range of species. However, the carry-on effects in adults of sublethal exposure occurring in larvae are not well characterized. Here, we use a collection of strains of Drosophila melanogaster differing in their larval resistance to a commonly used insecticide, imidacloprid, and we test the effect of larval exposure on behavioural traits at the adult stage. Focusing on locomotor activity and on courtship and mating behaviour, we observed a significant carry-on effect of imidacloprid exposure. The heritability of activity traits measured in flies exposed to imidacloprid was higher than measured in controls and in these, courtship traits were genetically less correlated from mating success. Altogether, we did not observe a significant effect of the larval insecticide resistance status on adult behavioural traits, suggesting that selection for resistance in larvae does not involve repeatable behavioural changes in adults. This lack of correlation between larval resistance and adult behaviour also suggests that resistance at the larval stage does not necessarily result in increased behavioural resilience at a later life stage. These findings imply that selection for resistance in larvae as well as for behavioural resilience to sublethal exposure in adult will combine and impose a greater evolutionary constraint. Our conclusions further substantiate the need to encompass multiple trait measures and life stages in toxicological assays to properly assess the environmental impact of pesticides.

Reconciliation of a gene network and species tree.

The phylogenetic trees of genes and the species which they belong to are similar, but distinct due to various evolutionary processes which affect genes but do not create new species. Reconciliations map the gene tree into the species tree, explaining the discrepancies by events including gene duplications and losses. However, when duplicate genes undergo recombination (a phenomenon known as paralog exchange, or non-allelic homologous recombination), the phylogeny of the genes becomes a network, not a tree. In this paper, we explore how to reconcile a gene network to a species tree with duplications and losses. We propose an extension of the lowest common ancestor (LCA) mapping which solves the problem for tree-child gene networks, show that a restricted version of the problem is polynomial-time solvable and bounds the optimal position of each gene node in the full problem, and show that the full problem is fixed-parameter tractable in the level of the gene network. This provides a formal foundation for the development of efficient algorithms to solve this problem.

GWAlpha: genome-wide estimation of additive effects (alpha) based on trait quantile distribution from pool-sequencing experiments.

Motivation: Sequencing pools of individuals (Pool-Seq) is a cost-effective way to gain insight into the genetics of complex traits, but as yet no parametric method has been developed to both test for genetic effects and estimate their magnitude. Here, we propose GWAlpha, a flexible method to obtain parametric estimates of genetic effects genome-wide from Pool-Seq experiments. Results: We showed that GWAlpha powerfully replicates the results of Genome-Wide Association Studies (GWAS) from model organisms. We perform simulation studies that illustrate the effect on power of sample size and number of pools and test the method on different experimental data. Availability and Implementation: GWAlpha is implemented in python, designed to run on Linux operating system and tested on Mac OS. It is freely available at https://github.com/aflevel/GWAlpha . Contact: afournier@unimelb.edu.au. Supplementary information: Supplementary data are available at Bioinformatics online.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala(301) to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala(301) to Ser resistance mutation and Met(360) to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser(301) homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser(301) change into an Ala(301) background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Dissecting the insect metabolic machinery using twin ion mass spectrometry: a single P450 enzyme metabolizing the insecticide imidacloprid in vivo.

Insecticide resistance is one of the most prevalent examples of anthropogenic genetic change, yet our understanding of metabolic-based resistance remains limited by the analytical challenges associated with rapidly tracking the in vivo metabolites of insecticides at nonlethal doses. Here, using twin ion mass spectrometry analysis of the extracts of whole Drosophila larvae and excreta, we show that (i) eight metabolites of the neonicotinoid insecticide, imidacloprid, can be detected when formed by susceptible larval genotypes and (ii) the specific overtranscription of a single gene product, Cyp6g1, associated with the metabolic resistance to neonicotinoids, results in a significant increase in the formation of three imidacloprid metabolites that are formed in C-H bond activation reactions; that is, Cyp6g1 is directly linked to the enhanced metabolism of imidacloprid in vivo. These results establish a rapid and sensitive method for dissecting the metabolic machinery of insects by directly linking single gene products to insecticide metabolism.

Small bowel tumors: pathology and management.

Despite comprising at least 75% of the length of the gastrointestinal tract, the small bowel only accounts for 3 to 6% of all its neoplasms. Forty different tumor subtypes arise from the small bowel; the commonest is adenoma, and malignant lesions include gastrointestinal stromal tumor neuroendocrine tumor lymphoma, and adenocarcinoma. Small bowel tumors typically cause either non-specific symptoms or none at all, which explains both the frequent delay in diagnosis and the wide range of potential investigations. The relative inaccessibility of the small bowel to endoscopic assessment is being challenged by the increased use of both capsule and double balloon endoscopy. Advances in endoscopic assessment are mirrored by improved sensitivity of radiological and nuclear imaging. Operative resection provides the mainstay of treatment for malignant disease (and symptomatic benign lesions), with oncological agents and somatostatin analogues providing useful adjuncts for inhibiting tumor growth and relieving symptoms. Survival reflects underlying tumor subtype, but is generally poor for malignant disease.

Phylogenomics of the Ecdysteroid Kinase-like (EcKL) Gene Family in Insects Highlights Roles in Both Steroid Hormone Metabolism and Detoxification.

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid molting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown, and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and we also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. Our evolutionary analyses provide hypotheses of function and a robust framework for future experimental studies of the EcKL gene family. They also open promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied coevolution of butterflies with their host plants.

Acetylcholine esterase of Drosophila melanogaster: a laboratory model to explore insecticide susceptibility gene drives.

BACKGROUND: One of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses. RESULTS: We have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygous, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of gene drive resistance (GDR): rather than being mediated by the conventional non-homologous end-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas. CONCLUSIONS: Insecticide susceptibility gene drives could be useful tools to control pest insects however problems with particularities of target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as a 'safety-switch' to prevent the unwanted spread of gene drives. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The haplolethal gene wupA of Drosophila exhibits potential as a target for an X-poisoning gene drive.

A synthetic gene drive that targets haplolethal genes on the X chromosome can skew the sex ratio toward males. Like an "X-shredder," it does not involve "homing," and that has advantages including the reduction of gene drive resistance allele formation. We examine this "X-poisoning" strategy by targeting 4 of the 11 known X-linked haplolethal/haplosterile genes of Drosophila melanogaster with CRISPR/Cas9. We find that targeting the wupA gene during spermatogenesis skews the sex ratio so fewer than 14% of progeny are daughters. That is unless we cross the mutagenic males to X^XY female flies that bear attached-X chromosomes, which reverses the inheritance of the poisoned X chromosome so that sons inherit it from their father, in which case only 2% of the progeny are sons. These sex ratio biases suggest that most of the CRISPR/Cas9 mutants we induced in the wupA gene are haplolethal but some are recessive lethal. The males generating wupA mutants do not suffer from reduced fertility; rather, the haplolethal mutants arrest development in the late stages of embryogenesis well after fertilized eggs have been laid. This provides a distinct advantage over genetic manipulation strategies involving sterility which can be countered by the remating of females. We also find that wupA mutants that destroy the nuclear localization signal of shorter isoforms are not haplolethal as long as the open reading frame remains intact. Like D. melanogaster, wupA orthologs of Drosophila suzukii and Anopheles mosquitos are found on X chromosomes making wupA a viable X-poisoning target in multiple species.

Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus.

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for approximately 16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession.

Rethinking the ecdysteroid source during Drosophila pupal-adult development.

Ecdysteroids, typified by 20-hydroxyecdysone (20E), are essential hormones for the development, reproduction and physiology of insects and other arthropods. For over half a century, the vinegar fly Drosophila melanogaster (Ephydroidea: Diptera) has been used as a model of ecdysteroid biology. Many aspects of the biosynthesis and regulation of ecdysteroids in this species are understood at the molecular level, particularly with respect to their secretion from the prothoracic gland (PG) cells of the ring gland, widely considered the dominant biosynthetic tissue during development. Discrete pulses of 20E orchestrate transitions during the D. melanogaster life cycle, the sources of which are generally well understood, apart from the large 20E pulse at the onset of pharate adult development, which has received little recent attention. As the source of this pharate adult pulse (PAP) is a curious blind spot in Drosophila endocrinology, we evaluate published biochemical and genetic data as they pertain to three hypotheses for the source of PAP 20E: the PG; an alternative biosynthetic tissue; or the recycling of stored 20E. Based on multiple lines of evidence, we contend the PAP cannot be derived from biosynthesis, with other data consistent with D. melanogaster able to recycle ecdysteroids before and during metamorphosis. Published data also suggest the PAP is conserved across Diptera, with evidence for pupal-adult ecdysteroid recycling occurring in other cyclorrhaphan flies. Further experimental work is required to test the ecdysteroid recycling hypothesis, which would establish fundamental knowledge of the function, regulation, and evolution of metamorphic hormones in dipterans and other insects.

SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans.

Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypovirulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely related CnSEC14-1 homologue, and CnSFH5, a distantly related SEC fourteen like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis.

A next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to Poa grasses.

BACKGROUND: Polyploidy is important from a phylogenetic perspective because of its immense past impact on evolution and its potential future impact on diversification, survival and adaptation, especially in plants. Molecular population genetics studies of polyploid organisms have been difficult because of problems in sequencing multiple-copy nuclear genes using Sanger sequencing. This paper describes a method for sequencing a barcoded mixture of targeted gene regions using next-generation sequencing methods to overcome these problems. RESULTS: Using 64 3-bp barcodes, we successfully sequenced three chloroplast and two nuclear gene regions (each of which contained two gene copies with up to two alleles per individual) in a total of 60 individuals across 11 species of Australian Poa grasses. This method had high replicability, a low sequencing error rate (after appropriate quality control) and a low rate of missing data. Eighty-eight percent of the 320 gene/individual combinations produced sequence reads, and >80% of individuals produced sufficient reads to detect all four possible nuclear alleles of the homeologous nuclear loci with 95% probability.We applied this method to a group of sympatric Australian alpine Poa species, which we discovered to share an allopolyploid ancestor with a group of American Poa species. All markers revealed extensive allele sharing among the Australian species and so we recommend that the current taxonomy be re-examined. We also detected hypermutation in the trnH-psbA marker, suggesting it should not be used as a land plant barcode region. Some markers indicated differentiation between Tasmanian and mainland samples. Significant positive spatial genetic structure was detected at <100 km with chloroplast but not nuclear markers, which may be a result of restricted seed flow and long-distance pollen flow in this wind-pollinated group. CONCLUSIONS: Our results demonstrate that 454 sequencing of barcoded amplicon mixtures can be used to reliably sample all alleles of homeologous loci in polyploid species and successfully investigate phylogenetic relationships among species, as well as to investigate phylogeographic hypotheses. This next-generation sequencing method is more affordable than and at least as reliable as bacterial cloning. It could be applied to any experiment involving sequencing of amplicon mixtures.

Ecdysteroid kinase-like (EcKL) paralogs confer developmental tolerance to caffeine in Drosophila melanogaster.

A unique aspect of metabolic detoxification in insects compared to other animals is the presence of xenobiotic phosphorylation, about which little is currently understood. Our previous work raised the hypothesis that members of the taxonomically restricted ecdysteroid kinase-like (EcKL) gene family encode the enzymes responsible for xenobiotic phosphorylation in the model insect Drosophila melanogaster (Diptera: Ephydroidea)-however, candidate detoxification genes identified in the EcKL family have yet to be functionally validated. Here, we test the hypothesis that EcKL genes in the rapidly evolving Dro5 clade are involved in the detoxification of plant and fungal toxins in D. melanogaster. The mining and reanalysis of existing data indicated multiple Dro5 genes are transcriptionally induced by the plant alkaloid caffeine and that adult caffeine susceptibility is associated with a novel naturally occurring indel in CG31370 (Dro5-8) in the Drosophila Genetic Reference Panel (DGRP). CRISPR-Cas9 mutagenesis of five Dro5 EcKLs substantially decreased developmental tolerance of caffeine, while individual overexpression of two of these genes-CG31300 (Dro5-1) and CG13659 (Dro5-7)-in detoxification-related tissues increased developmental tolerance. In addition, we found Dro5 loss-of-function animals also have decreased developmental tolerance of the fungal secondary metabolite kojic acid. Taken together, this work provides the first compelling functional evidence that EcKLs encode detoxification enzymes and suggests that EcKLs in the Dro5 clade are involved in the metabolism of multiple ecologically relevant toxins in D. melanogaster. We also propose a biochemical hypothesis for EcKL involvement in caffeine detoxification and highlight the many unknown aspects of caffeine metabolism in D. melanogaster and other insects.

Population genomics provides insights into lineage divergence and local adaptation within the cotton bollworm.

The cotton bollworm Helicoverpa armigera is a cosmopolitan pest and its diverse habitats plausibly contribute to the formation of diverse lineages. Despite the significant threat it poses to economic crops worldwide, its evolutionary history and genetic basis of local adaptation are poorly understood. In this study, we de novo assembled a high-quality chromosome-level reference genome of H. a. armigera (contig N50 = 7.34 Mb), with 99.13% of the HaSCD2 assembly assigned to 31 chromosomes (Z-chromosome + 30 autosomes). We constructed an ultradense variation map across 14 cotton bollworm populations and identified a novel lineage in northwestern China. Historical inference showed that effective population size changes coincided with global temperature fluctuation. We identified nine differentiated genes in the three H. armigera lineages (H. a. armigera, H. a. conferta and the new northwestern Chinese lineage), of which per and clk genes are involved in circadian rhythm. Selective sweep analyses identified a series of Gene Ontology categories related to climate adaptation, feeding behaviour and insecticide tolerance. Our findings reveal fundamental knowledge of the local adaptation of different cotton bollworm lineages and will guide the formulation of cotton bollworm management measures at different scales.

Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

Fighting fly genes.

Fighting by organisms such as mice and Drosophila provides model systems for investigating the genetic basis of aggression. Recent experiments to dissect male aggressive behaviour in Drosophila melanogaster, using gene expression analysis of selected lines followed by mutant analysis, have identified new candidate genes associated with male aggression, including one strong candidate that encodes a cytochrome P450 enzyme. Here, we describe the study of aggressive behaviour in flies and explore the possibility that cytochrome P450 is involved in aggression.