Toxicology and cytogenetic analysis of a Drosophila melanogaster mutant resistant to Imidacloprid and DDT

Resistance to all major insecticide classes has developed in numerous and diverse insect field populations. Imidacloprid, the worldwide most used neonicotinoid, has been extensively applied during the last decade for the control of different insect pests. Lately, cases of sporadic resistance also to neonicotinoids, including Imidacloprid, have been reported. Drosophila melanogaster is one of the most popular model organisms in biology and, although not a pest species, a promising model system for insecticide resistance research. In this study, we present a toxicological and karyotypic analysis of a Drosophila mutant (MiT[w-]3R2) resistant to Imidacloprid and cross-resistant to DDT. Karyotype analysis of polytene chromosome of MiT[w-]3R2 flies did not identify any apparent structural change of the polytene chromosome linked with the resistance phenotype

High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants

ModelPlex: Verified Runtime Validation of Verified Cyber-Physical System Models

Abstract. Formal verification and validation play a crucial role in making cyber-physical systems (CPS) safe. Formal methods make strong guarantees about the system behavior if accurate models of the system can be obtained, including mod-els of the controller and of the physical dynamics. In CPS, models are essential; but any model we could possibly build necessarily deviates from the real world. If the real system fits to the model, its behavior is guaranteed to satisfy the correct-ness properties verified w.r.t. the model. Otherwise, all bets are off. This paper introduces ModelPlex, a method ensuring that verification results about models apply to CPS implementations. ModelPlex provides correctness guarantees for CPS executions at runtime: it combines offline verification of CPS models with runtime validation of system executions for compliance with the model. Model-Plex ensures that the verification results obtained for the model apply to the ac-tual system runs by monitoring the behavior of the world for compliance with the model, assuming the system dynamics deviation is bounded. If, at some point, the observed behavior no longer complies with the model so that offline verifica-tion results no longer apply, ModelPlex initiates provably safe fallback actions. This paper, furthermore, develops a systematic technique to synthesize provably correct monitors automatically from CPS proofs in differential dynamic logic.

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

CRISPR/Cas-mediated gene editing using purified protein in Drosophila suzukii

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is an invasive pest of stone fruits and berries currently without effective control management. The sterile insect technique (SIT) is an environmentally friendly and effective pest control method that releases sterile males to mate with wild females resulting in the suppression or eradication of targeted pest populations. New molecular technologies and genome editing methods are paving the way to bring this technology to invasive species like D. suzukii. The CRISPR/Cas technology is emerging as a versatile option for efficient tailored gene manipulation. Components of this system, the guide RNA (gRNA) and Cas9 protein, can be delivered into the organism in the form of DNA, RNA, or protein. We report CRISPR/Cas-mediated site-specific white gene editing using purified Cas9 protein delivered directly into D. suzukii embryos. Mutant flies with 13, 3, and 1 nt deletions within the cutting s ite of the Cas9 protein were retrieved. Moreover, the two independent G1 deletion events (13 and 3 nt) derived from the same G0 female. Our results show that the recombinant Cas9 protein could be a method of choice for generating heritable gene modifications in D. suzukii

Imidacloprid does not induce Cyp genes involved in insecticide resistance of a mutant Drosophila melanogaster line

Certain xenobiotics have the capacity to induce the expression of genes involved in various biological phenomena, including insecticide resistance. The induction potential of different chemicals, among them different insecticides, has been documented for a number of insect species. In this study, we have analyzed the induction potential of Imidacloprid, a widely used member of the neonicotinoid insecticide family. Genes Cyp6g1 and Cyp6a2, known to be involved in the resistance of mutant Drosophila melanogaster line MiT[W]3R2 to Imidacloprid and DDT were included in the analyzed sample. We find that Imidacloprid does not induce expression of the analyzed genes

The effect of lead on fitness components and developmental stability in Drosophila subobscura

We analyzed the developmental time, egg-to-adult viability, and developmental stability (fluctuating wing size asymmetry) in Drosophila subobscura , maintained for six generations on different concentrations of lead. Development time is significantly affected by generation and lead concentration , but interaction of these factors is not a significant source of variability for this fitness component. Generation and the interaction generation × concentration of lead significantly affect egg-to-adult viability. Levene’s test of heterogeneity of variance showed that variability of FA is not significant in any of the samples. Within both lead concentrations females showed significantly higher FA indices for the wing width than males. Within sexes, a significantly higher FA was found only in females for wing width FA between the control and the lower concentration of lead. The results show that if strong relationship between FA and the studied fitness components exists, it results in a stronger selection of unstable genotypes under lead as a stress factor and, consequently, FA needs to be used with caution as a biomarker in natural populations under environmental stress

Organization and Product Design Pairings: A Review of Product Innovation Capabilities, Conceptualization and Future Directions

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Drawing on the Resource-based view of firms and market orientation theory, this paper presents a complementary view of the influence of product innovation capabilities (PICs) on organization and product design pairings. By exploring the influence of three complementary factors; market orientation, organizational form, and managerial strategic decisions, as essential determinants for emergence of architectural pairings, we theorize that PICs lead to better; organization and product design pairings selection, unique customer benefits, market acceptance, significant cost reductions, and new products development, and consequently superior organizational performance. We propose in essence, that PICs significantly shape and determine the success of architectural pairings between organization and product designs, and act as a source of sustainable competitive advantage for organizations. We further identify scope for future research to evaluate measures and validity of product innovation capability dimensions, and the extent of influence of the identified factors on architectural pairings