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Rapid dissemination of plasmid-borne polymyxin resistance mcr-1 genes threatens the efficacy of polymyxins. Acquisition of mcr-1 imposes a fitness cost on bacteria; identifying the molecular mechanisms underpinning this fitness cost will help in the development of adjunctive antimicrobial therapies that target polymyxin-resistant Gram-negative pathogens. Phenotypic assays and transcriptomics were acquired to investigate the impact of mcr-1 expression on membrane characteristics and transcriptomic responses in Escherichia coli TOP10 carrying the empty vector pBAD (TOP10+pBAD) and harbouring pBAD- mcr-1 (TOP10+pBAD- mcr-1). The overexpression of mcr-1 increased outer membrane permeability and caused membrane depolarisation, reflective of the transcriptomic results that showed downregulation of multiple genes involved in lipopolysaccharide core and O -antigen biosynthesis. Overexpression of mcr-1 also caused considerable gene expression changes in pathways involving carbohydrate metabolism (phosphotransferase system, pentose phosphate pathway, and pantothenate and coenzyme A biosynthesis), ABC transporters and intracellular responses to stress, especially those associated with oxidative and nucleic acid damage. Expression of mcr-1 also triggered the production of reactive oxygen species. These findings indicate that overexpression of mcr-1 results in persistent transcriptomic changes that primarily involve disruption to cell envelope synthesis via the reduction of LPS modifications, as well as dysregulation of carbon metabolism, redox balance and nucleic acids. These consequences of expression dysregulation may act as the main factors that impose a fitness cost with mcr-1 expression. [ABSTRACT FROM AUTHOR]

• Chat knockdown severely affected larval development and pupal eclosion, but vacht RNAi only disrupted pupal eclosion. • Parental RNAi of chat or vacht would reduce egg production and completely inhibite egg hatchability in T. castaneum. • Reduction of chat or vacht decreased the resistance to carbofuran and dichlorvos. Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are essential enzymes for synthesizing and transporting acetylcholine (ACh). But their functions in metamorphosis, reproduction, and the insecticide susceptibility were poorly understood in the insects. To address these issues, we identified the orthologues of chat and vacht in Tribolium castaneum. Spatiotemporal expression profiling showed Chat has the highest expression at the early adult stage, while vacht shows peak expression at the early larval stage. Both of them were highly expressed at the head of late adult. RNA interference (RNAi) of chat and vacht both led to a decrease in ACh content at the late larval stage. It is observed that chat knockdown severely affected larval development and pupal eclosion, but vacht RNAi only disrupted pupal eclosion. Further, parental RNAi of chat or vacht led to 35 % or 30 % reduction in fecundity, respectively, and knockdown of them completely inhibited egg hatchability. Further analysis has confirmed that both the reduction in fecundity and hatchability caused through the maternal specificity in T. castaneum. Moreover, the transcript levels of chat and vacht were elevated after carbofuran or dichlorvos treatment. Reduction of chat or vacht decreased the resistance to carbofuran and dichlorvos. This study provides the evidence for chat and vacht not only involved in development and reproduction of insects but also could as the potential targets of insecticides. [ABSTRACT FROM AUTHOR]

Myosin Myo20 plays vital roles in the morphogenesis of wings and legs among insects, but the function and signalling of Myo20 remain unclear. We show that Myo20 regulates wing cell division, ecdysteroid and amino acid metabolism, and gene expression in Tribolium castaneum. By RNA-seq, we identified 582 differentially expressed genes (DEGs) between control and ds- Myo20 larvae of T. castaneum. Of these DEGs, silencing Myo20 significantly decreased the mRNA and protein levels of lowfat. During development, lowfat has the highest expression in early pupae and the lowest level in 1-day embryos. Tissue-specific analysis indicated that lowfat was abundantly expressed in the head, fat body and epidermis of late-stage larvae and in wings and legs of 1, 2 and 5-day pupae. Likewise, knockdown of lowfat affected wing and leg morphogenesis, ecdysteroid and amino acid metabolism, and gene expression in T. castaneum. Silencing Myo20 or lowfat activated CYP18A1 to degrade ecdysteroids, stimulated amino acids catabolism to increase the transcription of 4E-BP but reduce S6K and cycE expression. These results suggest that Lowfat works downstream of Myo20 to employ target of rapamycin (TOR) signalling for wing and leg morphogenesis in insects. [Display omitted] • Myo20 regulates wing cell proliferation, ecdysteroid and amino acid metabolism, and gene expression. • Lowfat functions downstream of Myo20. • Myo20 and lowfat work upstream of TOR signalling to regulate wing morphogenesis. [ABSTRACT FROM AUTHOR]