Background: Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome). Results: To study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome. Conclusion: The current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.

Bacterial intracellular symbiosis (endosymbiosis) is widespread in nature and impacts many biological processes. In holometabolous symbiotic insects, metamorphosis entails a complete and abrupt internal reorganization that creates a constraint for endosymbiont transmission from larvae to adults. To assess how endosymbiosis copes—and potentially evolves—throughout this major host-tissue reorganization, we used the association between the cereal weevil Sitophilus oryzae and the bacterium Sodalis pierantonius as a model system. S. pierantonius are contained inside specialized host cells, the bacteriocytes, that group into an organ, the bacteriome. Cereal weevils require metabolic inputs from their endosymbiont, particularly during adult cuticle synthesis, when endosymbiont load increases dramatically. By combining dual RNA-sequencing analyses and cell imaging, we show that the larval bacteriome dissociates at the onset of metamorphosis and releases bacteriocytes that undergo endosymbiosis-dependent transcriptomic changes affecting cell motility, cell adhesion, and cytoskeleton organization. Remarkably, bacteriocytes turn into spindle cells and migrate along the midgut epithelium, thereby conveying endosymbionts to midgut sites where future mesenteric caeca will develop. Concomitantly, endosymbiont genes encoding a type III secretion system and a flagellum apparatus are transiently up-regulated while endosymbionts infect putative stem cells and enter their nuclei. Infected cells then turn into new differentiated bacteriocytes and form multiple new bacteriomes in adults. These findings show that endosymbiosis reorganization in a holometabolous insect relies on a synchronized host–symbiont molecular and cellular “choreography” and illustrates an adaptive feature that promotes bacteriome multiplication to match increased metabolic requirements in emerging adults. Significance Virtually all animals are associated with symbiotic bacteria. How these associations are modulated across an animal’s life cycle is a key question in understanding animal–bacteria interactions, particularly in organisms that undergo metamorphosis during development. Here, we used the cereal weevil to show how symbiosis is reorganized during metamorphosis, a developmental process entailing drastic tissue rearrangements. In this insect, symbionts are housed within specialized host cells, the bacteriocytes, that form the bacteriome tissue. We show that the bacteriome is completely remodeled during metamorphosis through host–symbiont communication, involving adhesion and motility host proteins and a temporary symbiont infectious behavior. This interkingdom dialogue results in the adaptation of the bacteriome to adulthood, highlighting the intertwining of symbiosis with host development.

Background: The rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions. Results: We sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino acids required for insect development and cuticle biosynthesis. Conclusions: Here we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes.

Background: Although native to North America, the invasion of the aphid-like grape phylloxeraDaktulosphaira vitifoliaeacross the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North AmericanVitisspecies as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome ofD. vitifoliaeas a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture.Results: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world.Conclusions: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently.

Insects subsisting on nutritionally unbalanced diets have evolved long-term mutualistic relationships with intracellular symbiotic bacteria (endosymbionts). The endosymbiont population load undergoes changes along with insect development. In the cereal weevil Sitophilus oryzae, the midgut endosymbionts Sodalis pierantonius drastically multiply following adult metamorphosis and rapidly decline until total elimination when the insect achieves its cuticle synthesis. Whilst symbiont load was shown to timely meet insect metabolic needs, little is known about the host molecular and immune processes underlying this dynamics. We performed RNA sequencing analysis on weevil midguts at three representative phases of the endosymbiont dynamics (i.e. increase, climax and decrease). To screen genes which transcriptional changes are specifically related to symbiont dynamics and not to the intrinsic development of the midgut, we further have monitored by RT-qPCR sixteen gene transcript levels in symbiotic and artificially non-symbiotic (aposymbiotic) weevils. We also localized the endosymbionts during the elimination process by fluorescence microscopy. Functional analysis of the host differentially expressed genes by RNA sequencing showed that the main transcriptional changes occur during endosymbiont growth phase and affect cell proliferation, apoptosis, autophagy, phagocytosis, and metabolism of fatty acids and nucleic acids. We also showed that symbiont dynamics alters the expression of several genes involved in insect development. Our results strengthened the implication of apoptosis and autophagy processes in symbiont elimination and recycling. Remarkably, apart from the coleoptericin A that is known to target endosymbionts and controls their division and location, no gene coding antimicrobial peptide was upregulated during the symbiont growth and elimination phases. We show that endosymbiont dynamics parallels numerous transcriptional changes in weevil developing adults and affects several biological processes, including metabolism and development. It also triggers cell apoptosis, autophagy and gut epithelial cell swelling and delamination. Strikingly, immunity is repressed during the whole process, presumably avoiding tissue inflammation and allowing insects to optimize nutrient recovery from recycled endosymbiont.