RESUMEN
The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.
Asunto(s)
Calliphoridae , Genoma Mitocondrial , Filogenia , Animales , Calliphoridae/genética , Northern Territory , Miasis/veterinaria , Miasis/parasitología , Miasis/genética , ARN de Transferencia/genética , ARN Ribosómico/genética , Dípteros/genética , Ovinos/parasitología , Ovinos/genéticaRESUMEN
Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.
Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Drosophila melanogaster/genética , Sueño/genética , Sueño REM , Encéfalo , MamíferosRESUMEN
BACKGROUND: Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. METHODS: In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. RESULTS: Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure, and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts-77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between Lucilia cuprina cuprina, L. c. dorsalis and L. sericata. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata. The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. CONCLUSIONS: Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia.
Asunto(s)
Dípteros , Miasis , Animales , Ovinos , Calliphoridae , Filogenia , Dípteros/genética , Miasis/epidemiología , Miasis/veterinaria , Genotipo , Victoria , Nucleótidos , GenómicaRESUMEN
Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, Gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.
RESUMEN
The nicotinic acetylcholine receptor (nAChR) subunit gene family consists of ten members in Drosophila melanogaster. The mature nAChR is a pentamer assembled from these subunits. Despite recent advances in the in vitro expression of some receptor subunit combinations (nAChR subtypes), the in vivo combinations and stoichiometry of these subtypes remains poorly defined. In addition, there are many potential nAChR signalling roles for different subtypes in insect behaviour, development and physiology. Prior work has shown that nAChR subunit mutants can display altered sleep and mating behaviour, disrupted hormone signalling and reduced locomotion, climbing ability and longevity. Teasing out the specific receptor subunits that are involved in these different functions is potentially made more difficult given that the structural similarity between members of gene families often means that there is a degree of functional redundancy. In order to circumvent this, we created a dual knockout strain for the Dα1 and Dß2 nAChR subunit genes and examined four traits including insecticide resistance. These subunits had been previously implicated in the response to a neonicotinoid insecticide, imidacloprid. The use of the dual knockout revealed that Dα1 and Dß2 subunits are involved in signalling that leads to the inflation of wings following adult emergence from the pupal case. The Dß1 subunit had previously been implicated as a contributor to this function. The lack of a phenotype or low penetrance of the phenotype in the Dα1 and Dß2 single mutants compared to the dual knockout suggests that these subunits are, to some extent, functionally redundant. We also observed stronger reductions in climbing ability and longevity in the dual knockout. Our findings demonstrate that a dual knockout approach to examining members of the nAChR subunit gene family may increase the power of genetic approaches linking individual subunits and combinations thereof to particular biological functions. This approach will be valuable as the nAChRs are so widely expressed in the insect brain that they are likely to have many functions that hereto remain undetected.
Asunto(s)
Insecticidas , Receptores Nicotínicos , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Inactivación de Genes , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Receptores Nicotínicos/metabolismoRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Macrólidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Combinación de Medicamentos , Insecticidas/administración & dosificación , Insecticidas/farmacología , Macrólidos/administración & dosificaciónRESUMEN
Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dß1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dß1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dß1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dß1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dß1 for the wing expansion pathway. Dß1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dß1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.
Asunto(s)
Drosophila melanogaster , AnimalesRESUMEN
Under exposure to harmful environmental stresses, organisms exhibit a general stress response involving upregulation of the expression of heat shock proteins (HSPs) which is thought to be adaptive. Small heat shock proteins (sHSPs) are key components of this response, although shsp genes may have other essential roles in development. However, the upregulation of expression of a suite of genes under stress may not necessarily be evidence of an adaptive response to stress that involves those genes. To explore this issue, we used the CRISPR/Cas9 system to investigate pleiotropic effects of the hsp23 gene in Drosophila melanogaster. Transgenic flies carrying a pCFD5 plasmid containing sgRNAs were created to generate a complete knockout of the hsp23 gene. The transgenic line lacking hsp23 showed an increased hatch rate and no major fitness costs under an intermediate temperature used for culturing the flies. In addition, hsp23 knockout affected tolerance to hot and cold temperature extremes but in opposing directions; knockout flies had reduced tolerance to cold, but increased tolerance to heat. Despite this, hsp23 expression (in wild type flies) was increased under both hot and cold conditions. The hsp23 gene was required for heat hardening at the pupal stage, but not at the 1st-instar larval stage, even though the gene was upregulated in wild type controls at that life stage. The phenotypic effects of hsp23 were not compensated for by expression changes in other shsps. Our study shows that the fitness consequences of an hsp gene knockout depends on environmental conditions, with potential fitness benefits of gene loss even under conditions when the gene is normally upregulated.
Asunto(s)
Frío , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Aptitud Genética , Proteínas de Choque Térmico/genética , Calor , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Técnicas de Inactivación de Genes , Proteínas de Choque Térmico/metabolismo , Masculino , Pupa/crecimiento & desarrolloRESUMEN
Insecticides remain valuable tools for the control of insect pests that significantly impact human health and agriculture. A deeper understanding of insecticide targets is important in maintaining this control over pests. Our study systematically investigates the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, we were able to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dß1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dα6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in ß1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dß1 examined here provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dß1 as a key component of nAChRs targeted by both these insecticide classes. A comparison of resistance phenotypes found in this study to resistance reported in insect pests reveals a strong conservation of subunit targets across many different insect species and that mutations have been identified in most of the receptor subunits that our findings would predict to have the potential to confer resistance.
Asunto(s)
Drosophila melanogaster , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Receptores Nicotínicos , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Combinación de Medicamentos , Macrólidos/farmacología , Mutación , Neonicotinoides/farmacología , Piridinas/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Compuestos de Azufre/farmacologíaRESUMEN
BACKGROUND: Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in α6-like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the α6 subunit, using genetic tools available in D. melanogaster. RESULTS: Functional, fluorescently tagged Dα6 subunits (Dα6YFP ) were developed to allow observation of the protein in vivo. Larvae expressing Dα6YFP were exposed to a sub-lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in Dα6YFP within 20 min, indicating that degradation of the Dα6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS-341 reduced loss of Dα6YFP in response to spinosyn A at the 20-min time point to 6.35%. In addition, in vivo administration of PS-341 prior to spinosad exposure reduced the effect of spinosad on larval activity. CONCLUSION: Based on these data, we propose that exposure to spinosad leads to degradation of the α6-like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. © 2021 Society of Chemical Industry.
Asunto(s)
Productos Biológicos , Insecticidas , Animales , Drosophila melanogaster , Combinación de Medicamentos , Resistencia a los Insecticidas , Insecticidas/farmacología , Macrólidos , Complejo de la Endopetidasa ProteasomalRESUMEN
BACKGROUND: Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. METHODS: Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. RESULTS: A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). CONCLUSIONS: Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.
Asunto(s)
Dípteros/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Miasis/veterinaria , Enfermedades de las Ovejas/parasitología , Secuencia de Aminoácidos , Animales , Australia , Dípteros/química , Dípteros/crecimiento & desarrollo , Dípteros/metabolismo , Femenino , Amplificación de Genes , Proteínas de Insectos/metabolismo , Masculino , Miasis/parasitología , Filogenia , Dominios Proteicos , Alineación de Secuencia , OvinosRESUMEN
Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.
Asunto(s)
Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/fisiología , Insecticidas/toxicidad , Neonicotinoides/toxicidad , Neuronas/efectos de los fármacos , Nitrocompuestos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Calcio/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Imidazoles/análisis , Imidazoles/toxicidad , Insecticidas/análisis , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neonicotinoides/análisis , Neuronas/metabolismo , Nitrocompuestos/análisis , Estrés Oxidativo/efectos de los fármacosRESUMEN
Imidacloprid, the world's most used insecticide, has caused considerable controversy due to harmful effects on non-pest species and increasing evidence showing that insecticides have become the primary selective force in many insect species. The genetic response to insecticides is heterogeneous across populations and environments, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, have revealed a mixed genetic architecture to resistance involving major genes (Paramyosin and Nicotinic-Acetylcholine Receptor Alpha 3) and polygenes with a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicated enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations in which chromosomal inversions In(2 L)t, In(3 R)Mo and In(3 R)Payne harbour susceptibility alleles. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.
Asunto(s)
Drosophila melanogaster/fisiología , Resistencia a los Insecticidas/fisiología , Insecticidas , Neonicotinoides , Nitrocompuestos , Termotolerancia , Animales , Clima , Femenino , Estudio de Asociación del Genoma Completo , Receptores Nicotínicos/genética , Tropomiosina/genéticaRESUMEN
The vinegar fly, Drosophila melanogaster, has made an enormous contribution to our understanding of insecticide targets, metabolism and transport. This contribution has been enabled by the unmatched capacity to manipulate genes in D. melanogaster and the fact that lessons learn in this system have been applicable to pests, because of the evolutionary conservation of key genes, particularly those encoding targets. With the advent of the CRISPR-Cas9 gene editing technology, genes can now be manipulated in pest species, but this review points to advantages that are likely to keep D. melanogaster at the forefront of insecticide research.
Asunto(s)
Sistemas CRISPR-Cas , Insectos/efectos de los fármacos , Resistencia a los Insecticidas , Insecticidas/efectos adversos , Animales , Drosophila melanogaster , Modelos AnimalesRESUMEN
BACKGROUND: Nitenpyram is a member of the economically important neonicotinoid class of insecticides. The in vivo metabolism of nitenpyram is not well characterised, but cytochrome P450 activity is the major mechanism of resistance to neonicotinoids identified in insect pests, and P450s metabolise other neonicotinoids including imidacloprid. RESULTS: Here, we used the GAL4-UAS targeted expression system to direct RNA interference (RNAi) against the cytochrome P450 redox partners to interrupt P450 functions in specific tissues in Drosophila melanogaster. RNAi of the mitochondrial redox partner defective in the avoidance of repellents (dare) in the digestive tissues reduced nitenpyram mortality, suggesting an activation step in the metabolism of nitenpyram carried out by a mitochondrial P450. RNAi of the mitochondrial cytochrome P450 Cyp12a5, which is expressed in the digestive tissues, resulted in the same phenotype, and transgenic overexpression of Cyp12a5 increased nitenpyram sensitivity. CONCLUSION: These results suggest that in vivo metabolism of nitenpyram by the mitochondrial P450 CYP12A5 results in the formation of a product with higher toxicity than the parent compound. © 2018 Society of Chemical Industry.
Asunto(s)
Familia 12 del Citocromo P450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Expresión Génica , Insecticidas/metabolismo , Proteínas Mitocondriales/genética , Neonicotinoides/metabolismo , Animales , Familia 12 del Citocromo P450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Proteínas Mitocondriales/efectos de los fármacosRESUMEN
Nicotinic acetylcholine receptors (nAChRs) have vital functions in processes of neurotransmission that underpin key behaviors. These pentameric ligand-gated ion channels have been used as targets for insecticides that constitutively activate them, causing the death of insect pests. In examining a knockout of the Dα1 nAChR subunit gene, our study linked this one subunit with multiple traits. We were able to confirm previous work that had identified Dα1 as a target of the neonicotinoid class of insecticides. Further, we uncovered roles for the gene in influencing mating behavior and patterns of sleep. The knockout mutant was also observed to have a significant reduction in longevity. This study highlighted the severe fitness costs that appear to be associated with the loss of function of this gene in natural populations in the absence of insecticides targeting the Dα1 subunit. Such a fitness cost could explain why target site resistances to neonicotinoids in pest insect populations have been associated specific amino acid replacement mutations in nAChR subunits, rather than loss of function. That mutant phenotypes were observed for the two behaviors examined indicates that the functions of Dα1, and other nAChR subunits, need to be explored more broadly. It also remains to be established whether these phenotypes were due to loss of the Dα1 receptor and/or to compensatory changes in the expression levels of other nAChR subunits.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Resistencia a los Insecticidas , Receptores Nicotínicos/metabolismo , Animales , Drosophila melanogaster/crecimiento & desarrollo , Esperanza de Vida , Conducta Sexual Animal , SueñoRESUMEN
Flystrike, or cutaneous myiasis, is caused by blow fly larvae of the genus Lucilia. This disease is a major problem in countries with large sheep populations. In Australia, Lucilia cuprina (Wiedemann, 1830) is the principal fly involved in flystrike. While much research has been conducted on L. cuprina, including physical, chemical, immunological, genetic and biological investigations, the molecular biology of this fly is still poorly understood. The recent sequencing, assembly and annotation of the draft genome and analyses of selected transcriptomes of L. cuprina have given a first global glimpse of its molecular biology and insights into host-fly interactions, insecticide resistance genes and intervention targets. The present article introduces L. cuprina, flystrike and associated issues, details past control efforts and research foci, reviews salient aspects of the L. cuprina genome project and discusses how the new genomic and transcriptomic resources for this fly might accelerate fundamental molecular research of L. cuprina towards developing new methods for the treatment and control of flystrike.
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Dípteros/genética , Interacciones Huésped-Parásitos , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Miasis/terapia , Animales , Genoma de los Insectos , OvinosRESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Microbioma Gastrointestinal , Inactivación Metabólica/genética , Insecticidas/metabolismo , Neonicotinoides/metabolismo , Nitrocompuestos/metabolismo , Alelos , Animales , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Expresión Génica , Técnicas de Inactivación de Genes , Genotipo , Insecticidas/farmacocinética , Cinética , Larva , Espectrometría de Masas , Neonicotinoides/farmacocinética , Nitrocompuestos/farmacocinética , Especificidad de Órganos , Variantes FarmacogenómicasRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Resistencia a los Insecticidas/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Anabasina/farmacología , Animales , Femenino , Expresión Génica/efectos de los fármacos , Insecticidas/farmacología , Masculino , Receptores Nicotínicos/biosíntesisRESUMEN
In Drosophila melanogaster larvae, the ring gland (RG) is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum, and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the RG's broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval RGs has provided a fascinating insight into the diversity of developmental signaling in this organ. We have found strong enrichment of expression of two gene pathways not previously associated with the RG: immune response and fatty acid metabolism. We have also uncovered strong expression for many uncharacterized genes. Additionally, RNA interference against RG-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency, respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the RG in governing insect development.