Complete Mitochondrial Genome for Lucilia cuprina dorsalis (Diptera: Calliphoridae) from the Northern Territory, Australia.

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.

Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia.

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.

Dual nicotinic acetylcholine receptor subunit gene knockouts reveal limits to functional redundancy.

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.

Loss of the Dß1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.

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.

Role of nicotinic acetylcholine receptor subunits in the mode of action of neonicotinoid, sulfoximine and spinosyn insecticides in Drosophila melanogaster.

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.

Evidence for activation of nitenpyram by a mitochondrial cytochrome P450 in Drosophila melanogaster.

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.

Transcriptome Analysis of Drosophila melanogaster Third Instar Larval Ring Glands Points to Novel Functions and Uncovers a Cytochrome p450 Required for Development.

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.

Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

[Effects of different fibre diameter of polypyrrole after direct current stimulation on adhesion and proliferation of cells and bacteria].

OBJECTIVE: To study the effects of cells'and bacteria's adhesion and proliferation on different fiber diameters of polypyrrole coating with electricity. METHODS: Titanium coated with polypyrrole was divided into no electrical stimulation and stimulation groups, each group had 30-60 nm, 70- 100 nm, 130-170 nm diameters of the fiber. MC3T3 cells and Staphylococcus aureus (S. aureus) were inoculated on different fiber diameters of polypyrrole coating with and without electric stimulation. We gave the electrical stimulation group 100 mV for 1 h and every 24 hours gave it 1 h stimulation, and no electrical stimulation group was not managed. We used scanning electron microscope (SEM) to observe the cells'and bacteria's morphology. The cells were given 20 mL CCK-8 solutions after 1,3,7 days' cultivation, then incubated for 2 h, the solution was transferred to 96-well plate, we measured the cells' CCK-8 of the 30-60 nm, 70-100 nm, and 130-170 nm groups by Elisa. The cells on different fiber diameters were also stained by live-dead cell staining kit, TritonX-100 and DAPI. We used PBS to wash and glycerin to seal them. The live-dead situation and morphology were tested by co focal microscope. The bacterial were stained by Live/dead baclight bacterial viability kits, we detect the suspension's D of the 30-60 nm, 70-100 nm, and 130-170 nm groups, and also observed the bacteria's survival situation by co focal microscope. RESULTS: The CCK-8 of the cells with direct current stimulation was higher than that of the unpowered group (F=12.248, P=0.006). The smaller the fiber diameter, the better was the cell's adhesion and proliferation (F=9.261, P=0.005). The bacterial suspension's D of the electric group was lower than that of the unpowered group, and the fiber diameter had no significant effect on the bacteria's growth(F=9.641, P=0.036). CONCLUSION: Polypyrrole coating with electricity can promote the cell's proliferation and inhibit the bacteria's proliferation, and the cell growth on small fiber diameter coating is better. There is no difference in the bacterial growth of different fiber diameter coatings.

Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction.

Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and their biology.

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster.

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.