microRNA maintains nutrient homeostasis in the symbiont-host interaction.

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control.

Characterization of carotenoid biosynthetic pathway genes in the pea aphid (Acyrthosiphon pisum) revealed by heterologous complementation and RNA interference assays.

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase, desaturase, and cyclase activities. In the pea aphid (Acyrthosiphon pisum), the duplication of carotenoid biosynthetic genes, including carotenoid synthases/cyclases (ApCscA-C) and desaturases (ApCdeA-D), through horizontal gene transfer from fungi has been detected, and ApCdeB has known dehydrogenation functions. However, whether other genes contribute to aphid carotenoid biosynthesis, and its specific regulatory pathway, remains unclear. In the current study, functional analyses of seven genes were performed using heterologous complementation and RNA interference assays. The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene, and ApCscC may also have a cyclase activity. ApCdeA, ApCdeC, and ApCdeD had diverse dehydrogenation functions. ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene (three-step product), ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene (two-step product), and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene (four-step product). Silencing of ApCscs reduced the expression levels of ApCdes, and silencing these carotenoid biosynthetic genes reduced the α-, ß-, and γ-carotene levels, as well as the total carotenoid level. The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

RNA-seq Analysis of Clitea metallica (Coleoptera: Chrysomelidae), Provides Insights Into Cuticle-Related Genes and miRNAs.

The citrus leaf beetle, Clitea metallica, is a specialized citrus pest through feeding on fresh leaves by larva and adults, and causes nicks and holes into leaves, leaving only a waxy surface layer. Insect cuticle is a complex exoskeleton that is not only involved in development but also protects the insect from environmental contaminations. Due to these key roles of the cuticle, cuticle-related genes are currently investigated in understanding the insect physiology in adaptation. Therefore, in this study, we built two libraries, transcriptomic (43 million clean reads) and small RNA (17 million clean reads), of C. metallica to identify cuticle-related genes and possibly associated miRNAs, being as an example to explore these data sets. Our results showed that a total of 47 cuticular protein genes were identified and most of these genes harbored a conserved motif (the Rebers and Riddiford motif) and belonged to the CPR family. Unigenes encoding proteins involved in chitin synthesis and degradation were also identified, including chitin synthase (2 unigenes), chitinase (14 unigenes), chitinase-like protein (2 unigenes), and chitin deacetylase (5 unigenes). Based on the small RNA library, we identified 30 miRNAs conserved across insect species. Among these miRNAs, 14 were predicted to be target genes associated with cuticle synthesis and degradation. In summary, 70 cuticle-related genes and 14 cuticle-related miRNAs were identified based on the transcriptome and small RNA library of C. metallica. These data sets will promote the understanding of cuticle molecular regulation in C. metallica as well as provide new potential targets for pest control.