Vertically transmitted endosymbionts persist for millions of years in invertebrates and play an important role in animal evolution. However, the functional basis underlying the maintenance of these long-term resident bacteria is unknown. We report that the weevil coleoptericin-A (ColA) antimicrobial peptide selectively targets endosymbionts within the bacteriocytes and regulates their growth through the inhibition of cell division. Silencing the colA gene with RNA interference resulted in a decrease in size of the giant filamentous endosymbionts, which escaped from the bacteriocytes and spread into insect tissues. Although this family of peptides is commonly linked with microbe clearance, this work shows that endosymbiosis benefits from ColA, suggesting that long-term host-symbiont coevolution might have shaped immune effectors for symbiont maintenance.

Symbiotic associations are widespread in nature and represent a driving force in evolution. They are known to impact fitness, and thereby shape the host phenotype. Insects subsisting on nutritionally poor substrates have evolved mutualistic relationships with intracellular symbiotic bacteria (endosymbionts) that supply them with metabolic components lacking in their diet. In many species, endosymbionts are hosted within specialized host cells, called the bacteriocytes, and transmitted vertically across host generations. How hosts balance the costs and benefits of having endosymbionts, and whether and how they adjust symbiont load to their physiological needs, remains largely unexplored. By investigating the cereal weevil Sitophilus association with the Sodalis pierantonius endosymbiont, we discover that endosymbiont populations intensively multiply in young adults, before being rapidly eliminated within few days. We show that young adults strongly depend on endosymbionts and that endosymbiont proliferation after metamorphosis matches a drastic host physiological need for the tyrosine (Tyr) and phenylalanine (Phe) amino acids to rapidly build their protective exoskeleton. Tyr and Phe are precursors of the dihydroxyphenylalanine (DOPA) molecule that is an essential component for the cuticle synthesis. Once the cuticle is achieved, DOPA reaches high amounts in insects, which triggers endosymbiont elimination. This elimination relies on apoptosis and autophagy activation, allowing digestion and recycling of the endosymbiont material. Thus, the weevil-endosymbiont association reveals an adaptive interplay between metabolic and cellular functions that minimizes the cost of symbiosis and speeds up the exoskeleton formation during a critical phase when emerging adults are especially vulnerable.

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Intracellular symbiosis is widespread in the insect world where it plays an important role in evolution and adaptation. The weevil family Dryophthoridae (Curculionoidea) is of particular interest in intracellular symbiosis evolution with regard to the great economical and ecological features of these invasive insects, and the potential for comparative studies across a wide range of host plants and environments. Here, we have analyzed the intracellular symbiotic bacteria of 19 Dryophthoridae species collected worldwide, representing a wide range of plant species and tissues. All except one (Sitophilus linearis) harbor symbiotic bacteria within specialized cells (the bacteriocytes) assembled as an organ, the bacteriome. Phylogenetic analysis of the 16S rDNA gene sequence of the Dryophthoridae endosymbionts revealed three endosymbiotic clades belonging to gamma3-Proteobacteria and characterized by different GC contents and evolutionary rate. The genus name Candidatus Nardonella was proposed for the ancestral clade infesting Dryophthoridae 100 MYA and represented by five of nine bacterial genera studied. For this clade showing low GC content (40.5% GC) and high evolutionary rate (0.128 substitutions/site per 100 Myr), a single infection and subsequent cospeciation of the host and the endosymbionts was observed. In the two other insect lineage endosymbionts, with relatively high GC content (53.4% and 53.8% GC), competition with ancestral pathogenic bacteria might have occurred, leading to endosymbiont replacement in present-day last insects.

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).

Bacterial intracellular symbiosis (endosymbiosis) is well documented in the insect world where it is believed to play a crucial role in adaptation and evolution. However, although Coleopteran insects are of huge ecological and economical interest, endosymbiont molecular analysis is limited to the Dryophthoridae family. Here, we have analyzed the intracellular symbiotic bacteria in 2 Hylobius species belonging to the Molytinae subfamily (Curculionoidea superfamily) that exhibit different features from the Dryophthoridae insects in terms of their ecology and geographical spanning. Fluorescence in situ hybridization has shown that both Hylobius species harbor rod-shaped pleiomorphic symbiotic bacteria in the oocyte and in the bacteria-bearing organ (the bacteriome), with a shape and location similar to those of the Dryophthoridae bacteriome. Phylogenetic analysis of the 16S ribosomal DNA gene sequences, using the heterogeneous model of DNA evolution, has placed the Hylobius spp. endosymbionts (H-group) at the basal position of the ancestral R-clade of Dryophthoridae endosymbionts named Candidatus Nardonella but relatively distant from the S-clade of Sitophilus spp. endosymbionts. Endosymbionts from the H-group and the R-clade evolved more quickly compared with free-living enteric bacteria and endosymbionts from the S- and D-clades of Dryophthoridae. They are AT biased (58.3% A + T), and they exhibit AT-rich insertions at the same position as previously described in the Candidatus Nardonella 16S rDNA sequence. Moreover, the host phylogenetic tree based on the mitochondrial COI gene was shown to be highly congruent with the H-group and the R-clade, the divergence of which was estimated to be around 125 MYA. These new molecular data show that endosymbiosis is old in Curculionids, going back at least to the common ancestor of Molytinae and Dryophthoridae, and is evolutionary stable, except in 2 Dryophthoridae clades, providing additional and independent supplementary evidence for endosymbiont replacement in these taxa.

Among the striking attributes of eukaryotic organisms is their ability to evolve close associations with diverse bacteria, with a continuum of phenotypes ranging from pathogenic to mutualistic (28,29). However, the molecular mechanisms and responses of the host immune system underlying a given phenotype remain poorly understood, particularly in invertebrate intracellular symbiosis. In insects, endosymbionts are maternally inherited, and early insect embryogenesis is marked by bacterial invasion and the subsequent differentiation of specialized host cells, called bacteriocytes, that often form an organ called the bacteriome (4, 18). Functions of the bacteriocyte cell include nutrient synthesis, molecular exchange between the host and the bacteria (12, 21), and presumably the control of endosymbionts and their isolation from the insect immune system. Insect intracellular symbiosis increases host fitness and invasive power (17) and, over evolutionary time, leads to a complete interdependence between the bacteria and the host. Due to evolutionary constraints associated with a strictly intracellular life style and maternal inheritance of the symbionts, selection pressure favors, during the host-symbiont coevolution, deletions of bacterial genes encoding redundant metabolic pathways with the host (34), DNA recombinational repair genes (11), and genes becoming "unnecessary" to the new association. Among the latter, virulence genes and genes encoding the bacterial cell wall elements have been prone to serial gene deletions, as evidenced in insect endosymbiont genomes sequenced so far (1,15,34). Hence, association features may rely not only on the host functions but also on the level of bacterial genomic alteration and, thereby, on the age of the association. More recent associations have fewer bacterial genomic alterations than their free-living relatives. In this evolutionary context, recent associations with less deleted bacterial genomes should provide insight into the early stages in the interplay between host immunity and bacterial virulence. Among insect endosymbioses, the Sitophilus species associations provide an excellent model for studying innate immune responses, because the association was established relatively recently (less than 25 million years ago), probably by endosymbiont replacement (26). The endosymbiont genomes have not experienced severe gene deletion (7), and these genomes encode functional secretion systems (10).Recent studies have uncovered a remarkable conservation in innate immune defense mechanisms among plants, insects, and mammals, which suggests a common ancestry of the system (6, 23). An initial step of the immune reaction is the perception of conserved microbial cell envelope motifs, such as peptidoglycan (PGN), through recognition receptors (22). Among these are the peptidoglycan recognition proteins (PGRP) that recognize the DAP type and/or the Lys type of PGN from gramnegative and gram-positive bacterial peptidoglycan, respectively (25,27). These evolutionarily conserved proteins con...

Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevilSitophilus zeamais’s association withSodalis pierantoniusendosymbiont. We discovered that weevilpgrp-lbgenerates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC–independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts.pgrp-lbsilencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereaspgrp-lbwas first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.