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.

An Approach for Selecting a Model for the Assessment of Potentially Contaminated Sites.

Assessment of potentially contaminated sites (PCS) can be expensive; hence, simple and less demanding methods and models are required. This work attempts to provide an approach that can aid in selecting the most appropriate model for the PCS. The developed method uses over 100 field site data to evaluate four test models (analytical/empirical) that provide the maximum plume length (Lmax ), which is used as a principal model ranking quantity in this work. Analysis of site data shows that field plume length (Lf ) follows a log-normal distribution. Subsequently, Lmax is delineated with respect to Lf using a threshold probability as underestimating, overestimating, and overly-overestimating. Akaike information criterion (AIC) and analytical hierarchy process (AHP) are considered to support the threshold approach results. The classical AIC is modified (to AICmod ) to fit the term represented by the difference between Lf and Lmax . Additionally, the threshold factors as a product of subjective weights are added to the AICmod . Using Lf and Lmax , the AICmod provides a distinct ranking of the test models. For the AHP approach, the goodness of fit, underestimation, overly overestimation, and model complexity are the four chosen criteria. Similar to AICmod , the AHP approach provides a distinct ranking of the test models. The final decision on the best fitting model can be made on user criteria following the scheme developed in this work.

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.

In vitro evaluation of the effect of different polishing techniques on the surface roughness of composite resins submitted to at-home and in-office bleaching procedures.

OBJECTIVE: Bleaching agents may affect the properties of dental materials. The aim of this study was to evaluate the effect of different polishing techniques on the surface roughness of composite resins submitted to the at-home and in-office bleaching treatment. MATERIALS AND METHODS: Disc-shaped specimens were carried out of nanofilled and microhybrid composites (n = 10). Finishing step was performed after light curing (L1) and polishing after 24 h with two systems (L2). Then, specimens were submitted to the home or in-office bleaching procedures, and roughness was re-evaluated (L3). The surface roughness (Ra) readings were measured at L1, L2, and L3 times using a profilometer. Data were statistically analyzed by multiple-way analysis of variance and Tukey test (α = 0.05). RESULTS: The polishing procedures decreased Ra for both composites compared to baseline values (L1). The roughness of specimens polished with jiffy did not present significant difference after polishing step (L2) and bleaching treatment (L3). However, the groups polished with Sof-Lex discs had increase on the Ra values after bleaching. CONCLUSION: The polishing is an important procedure to reduce the roughness of dental restorations and composite surface polished with jiffy system improved the degradation resistance to the bleaching agents compared to Sof-Lex discs.

Influence of flowable composite and restorative technique on microleakage of class II restorations.

AIM: The aim of the present study was to evaluate the microleakage in class II cavities restored with dental composite by varying the thickness of two flowable composite resins and the restorative technique. METHODS: A total of 130 cavities were prepared on proximal surfaces of bovine teeth and randomly divided in 13 groups. Twelve experimental groups were performed using flowable composite resin as the liner, and they were divided according to the type of flowable composite resin (Filtek Z350 Flow-Z350F and SureFil SDR Flow-SUR), the thickness of the flowable composite (0.5, 1, or 2 mm), and the restorative technique (flowable composite applied at the cavosurface angle or amelodentinal junction) (n = 10). A control group was performed without using a flowable composite. Following restorative procedures and thermocycling, the samples were immersed in Methylene Blue for 2 h, and prepared for analysis in an absorbance spectrophotometer. All results were statistically analyzed (α = 0.05). RESULTS: There was no significant difference between the flowable composites (P > 0.05). The restorative techniques influenced microleakage and were flowable composite dependent (P ≤ 0.05). The lower thickness of the flowable composite provided less microleakage (P ≤ 0.05). CONCLUSION: The use a lower thickness of a flowable composite resin provided less microleakage, suggesting a better sealing tooth-restoration interface.