Use of Mutagenesis, Genetic Mapping and Next Generation Transcriptomics to Investigate Insecticide Resistance Mechanisms

Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w−]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results

Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vector Culex quinquefasciatus

Culex quinquefasciatus plays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackle C. quinquefasciatus vectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, including C. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistant C. quinquefasciatus was explored to identify candidate genes associated with insecticide resistance. High levels of insecticide resistance were observed for five out of six insecticides tested, with the lowest mortality (0.97%) reported to permethrin, while for DDT, lambdacyhalothrin, bendiocarb and deltamethrin the mortality rate ranged from 1.63–3.29%. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1 h exposure and 58.02% after 4 h. Genotyping of the G119S Ace-1 target site mutation detected a highly significant association (p 8-fold increase vs unexposed controls). These results provide evidence that bendiocarb resistance in Ugandan C. quinquefasciatus is mediated by both target-site mechanisms and over-expression of detoxification enzymes

Antidepressants’ effects on testosterone and estrogens: What do we know?

Various antidepressants are commonly used to treat depression and anxiety disorders, and sex differences have been identified in their efficacy and side effects. Steroids, such as estrogens and testosterone, both in the periphery and locally in the brain, are regarded as important modulators of these sex differences. This review presents published data from preclinical and clinical studies that measure testosterone and estrogen level changes during and/or after acute or chronic administration of different antidepressants. The majority of studies show an interaction between sex hormones and antidepressants on sexual function and behavior, or in depressive symptom alleviation. However, most of the studies omit to investigate antidepressants’ effects on circulating levels of gonadal hormones. From data reviewed herein, it is evident that most antidepressants can influence testosterone and estrogen levels. Still, the evidence is conflicting with some studies showing an increase, others decrease or no effect. Most studies are conducted in male animals or humans, underscoring the importance of considering sex as an important variable in such investigations, especially as depression and anxiety disorders are more common in women than men. Therefore, research is needed to elucidate the extent to which antidepressants can influence both peripheral and brain levels of testosterone and estrogens, in males and females, and whether this impacts the effectiveness or side effects of antidepressants. © 2021 Elsevier B.V

Pharmacology of ketamine and esketamine as rapid-acting antidepressants

The lack of utter efficacy and fast action of commonly used antidepressants that selectively target the monoaminergic neurotransmission has led to the exploration of ketamine's actions. Ketamine's antidepressant effect was firstly described in 1973 and nowadays its therapeutic value as a fast- and long- lasting antidepressant has been extensively established. Ketamine is an antagonist of the N-Methyl-D-aspartate receptor (NMDAR) and its main mechanism of action via NMDAR inhibition expressed in GABAergic (gamma-Aminobutyric acid, GABA) interneurons may be relayed to its antidepressant effects. This review aims to describe the pharmacokinetic and pharmacodynamic profile of ketamine when used for treatment-resistant depression. Moreover, ketamine is a racemic mixture consisting of two enantiomers, R- and S- ketamine. We describe the pharmacology of esketamine, along with the guidelines for effective and safe intranasal administration of esketamine. Lastly, this review presents sex differences in preclinical and clinical studies of ketamine and esketamine administration

Spatial consistency for multiple source direction-of-arrival estimation and source counting.

A conventional approach to wideband multi-source (MS) direction-of-arrival (DOA) estimation is to perform single source (SS) DOA estimation in time-frequency (TF) bins for which a SS assumption is valid. The typical SS-validity confidence metrics analyse the validity of the SS assumption over a fixed-size TF region local to the TF bin. The performance of such methods degrades as the number of simultaneously active sources increases due to the associated decrease in the size of the TF regions where the SS assumption is valid. A SS-validity confidence metric is proposed that exploits a dynamic MS assumption over relatively larger TF regions. The proposed metric first clusters the initial DOA estimates (one per TF bin) and then uses the members' spatial consistency as well as its cluster's spread to weight each TF bin. Distance-based and density-based clustering are employed as two alternative approaches for clustering DOAs. A noise-robust density-based clustering is also used in an evolutionary framework to propose a method for source counting and source direction estimation. The evaluation results based on simulations and also with real recordings show that the proposed weighting strategy significantly improves the accuracy of source counting and MS DOA estimation compared to the state-of-the-art

Transgenic expression of the Aedes aegypti CYP9J28 confers pyrethroid resistance in Drosophila melanogaster

The emergence and spread of insecticide resistance in mosquitoes, such as the major vector of dengue and yellow fever Aedes aegypti, is a major public health problem. A number of studies have been conducted to-date aiming to identify specific molecular changes that are associated with the phenotype, in an attempt to use this knowledge to control the development and spread of resistant populations. Several approaches, ranging from classical techniques to high-throughput transcriptomics, have revealed a large number of genes which are associated with the trait. However, the development of functional tools for the validation of the possible role of those genes (if any) in mosquito resistance has not kept similar pace. Here, we report the use of Drosophila melanogaster as a model for establishing the role of A. aegypti cytochrome P450s. We showed that the transgenic expression of the CYP9J28 a gene which is upregulated in pyrethroid resistant A. aegypti mosquitoes and encodes an enzyme capable of metabolizing pyrethroids, in D. melanogaster confers significant levels of pyrethroid resistance. Our study provides solid functional in vivo evidence for the role of the AaegCYP9J28 in pyrethroid resistance and shows for the first time that ectopic expression of mosquito genes in D. melanogaster can be a robust approach for validating candidate resistance genes. Further potential applications of the approach are discussed

Age-related dysregulation of homeostatic control in neuronal microcircuits

Neuronal homeostasis prevents hyperactivity and hypoactivity. Age-related hyperactivity suggests homeostasis may be dysregulated in later life. However, plasticity mechanisms preventing age-related hyperactivity and their efficacy in later life are unclear. We identify the adult cortical plasticity response to elevated activity driven by sensory overstimulation, then test how plasticity changes with age. We use in vivo two-photon imaging of calcium-mediated cellular/synaptic activity, electrophysiology and c-Fos-activity tagging to show control of neuronal activity is dysregulated in the visual cortex in late adulthood. Specifically, in young adult cortex, mGluR5-dependent population-wide excitatory synaptic weakening and inhibitory synaptogenesis reduce cortical activity following overstimulation. In later life, these mechanisms are downregulated, so that overstimulation results in synaptic strengthening and elevated activity. We also find overstimulation disrupts cognition in older but not younger animals. We propose that specific plasticity mechanisms fail in later life dysregulating neuronal microcircuit homeostasis and that the age-related response to overstimulation can impact cognitive performance