Insects' intestinal microbes have profound effects on the host's physiological traits, which can impact their physiology at both the local (gut) and systemic (body) levels. Nevertheless, the molecular mechanisms underlying hostmicrobiota interactions, especially in non-model insects, remain elusive. Recently, tissue-specific transcriptomic analysis has been highlighted as a robust tool in studying host-microbe interactions. Plagiodera versicolora is a worldwide leaf-eating pest that primarily feeds on willows and poplar. The interplay between gut microflora and this host beetle has yet to be studied. Herein, we investigate the effects of the gut microbiota on the body mass of P. versicolora larvae, compare the nutrition status of larvae in absence and presence of gut microbiota, and profile gut bacterial loads throughout its developmental larval stages. We then perform comparative transcriptomic analysis of gut and body tissues in axenic and non-axenic larvae. Finally, we confirm the expression patterns of representative genes in nutritional metabolism and immunity. Results show that weight growth is retarded in conventional larvae, with a concomitant increase of total bacterial load by the 5 th development day, and germ-free larvae have a higher glucose content than conventional-reared larvae. Both nutritional and immunological analyses indicate that gut bacteria are a burden in the beetle's larval development. These findings elucidate the impacts of gut microbiota on P. versicolora, and provide insight into tissue-specific responses to gut microflora in this pest at the genetic level, boosting our understanding of the molecular mechanisms underlying host-microbe interactions in leaf beetles and beyond.

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

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Insects can sense surrounding chemical signals by their accurate chemosensory systems. This system plays a vital role in the life history of insects. Several gene families participate in chemosensory processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs), and sensory neuron membrane proteins (SNMPs). Plagiodera versicolora (Coleoptera: Chrysomelidae), is a leaf-eating forest pest found in salicaceous trees worldwide. In this study, a transcriptome analysis of male and female adult antennae in P. versicolora individuals was conducted, which identified a total of 98 candidate chemosensory genes including 40 ORs, 7 IRs, 13 GRs, 10 CSPs, 24 OBPs, and 4 SNMPs. Subsequently, the tissue expression profiles of 15 P. versicolora OBPs (PverOBPs) and 39 ORs (PverORs) were conducted by quantitative real-time PCR. The data showed that almost all PverOBPs and PverORs were highly expressed in the male and female antennae. In addition, several OBPs and ORs (PverOBP10, PverOBP12, PverOBP18, PverOR24, and PverOR35) had higher expression levels in female antennae than those in the male antennae, indicating that these genes may be taking part in some female-specific behaviors, such as find mates, oviposition site, etc. This study deeply promotes further understanding of the chemosensory system and functional studies of the chemoreception genes in P. versicolora.