Spatial and morphological reorganization of endosymbiosis during metamorphosis accommodates adult metabolic requirements in a weevil.

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.

Weevil pgrp-lb prevents endosymbiont TCT dissemination and chronic host systemic immune activation.

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 weevil Sitophilus zeamais's association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates 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-lb silencing 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. Whereas pgrp-lb was 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.

The transposable element-rich genome of the cereal pest Sitophilus oryzae.

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.

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.

What can a weevil teach a fly, and reciprocally? Interaction of host immune systems with endosymbionts in Glossina and Sitophilus.

The tsetse fly (Glossina genus) is the main vector of African trypanosomes, which are protozoan parasites that cause human and animal African trypanosomiases in Sub-Saharan Africa. In the frame of the IAEA/FAO program 'Enhancing Vector Refractoriness to Trypanosome Infection', in addition to the tsetse, the cereal weevil Sitophilus has been introduced as a comparative system with regards to immune interactions with endosymbionts. The cereal weevil is an agricultural pest that destroys a significant proportion of cereal stocks worldwide. Tsetse flies are associated with three symbiotic bacteria, the multifunctional obligate Wigglesworthia glossinidia, the facultative commensal Sodalis glossinidius and the parasitic Wolbachia. Cereal weevils house an obligatory nutritional symbiosis with the bacterium Sodalis pierantonius, and occasionally Wolbachia. Studying insect host-symbiont interactions is highly relevant both for understanding the evolution of symbiosis and for envisioning novel pest control strategies. In both insects, the long co-evolution between host and endosymbiont has led to a stringent integration of the host-bacteria partnership. These associations were facilitated by the development of specialized host traits, including symbiont-housing cells called bacteriocytes and specific immune features that enable both tolerance and control of the bacteria. In this review, we compare the tsetse and weevil model systems and compile the latest research findings regarding their biological and ecological similarities, how the immune system controls endosymbiont load and location, and how host-symbiont interactions impact developmental features including cuticle synthesis and immune system maturation. We focus mainly on the interactions between the obligate symbionts and their host's immune systems, a central theme in both model systems. Finally, we highlight how parallel studies on cereal weevils and tsetse flies led to mutual discoveries and stimulated research on each model, creating a pivotal example of scientific improvement through comparison between relatively distant models.

Weevil endosymbiont dynamics is associated with a clamping of immunity.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Development, feeding, and sex shape the relative quantity of the nutritional obligatory symbiont Wolbachia in bed bugs.

The common bed bug, Cimex lectularius, is a hemipteran insect that feeds only on blood, and whose bites cause public health issues. Due to globalization and resistance to insecticides, this pest has undergone a significant and global resurgence in recent decades. Blood is an unbalanced diet, lacking notably sufficient B vitamins. Like all strict hematophagous arthropods, bed bugs host a nutritional symbiont supplying B vitamins. In C. lectularius, this nutritional symbiont is the intracellular bacterium Wolbachia (wCle). It is located in specific symbiotic organs, the bacteriomes, as well as in ovaries. Experimental depletion of wCle has been shown to result in longer nymphal development and lower fecundity. These phenotypes were rescued by B vitamin supplementation. Understanding the interaction between wCle and the bed bug may help to develop new pest control methods targeting the disruption of this symbiotic interaction. The objective of this work was thus to quantify accurately the density of wCle over the life cycle of the host and to describe potential associated morphological changes in the bacteriome. We also sought to determine the impact of sex, feeding status, and aging on the bacterial population dynamics. We showed that the relative quantity of wCle continuously increases during bed bug development, while the relative size of the bacteriome remains stable. We also showed that adult females harbor more wCle than males and that wCle relative quantity decreases slightly in adults with age, except in weekly-fed males. These results are discussed in the context of bed bug ecology and will help to define critical points of the symbiotic interaction during the bed bug life cycle.

Endosymbiont-containing germarium transcriptional survey in a cereal weevil depicts downregulation of immune effectors at the onset of sexual maturity.

Insects often establish long-term relationships with intracellular symbiotic bacteria, i.e., endosymbionts, that provide them with essential nutrients such as amino acids and vitamins. Endosymbionts are typically confined within specialized host cells called bacteriocytes that may form an organ, the bacteriome. Compartmentalization within host cells is paramount for protecting the endosymbionts and also avoiding chronic activation of the host immune system. In the cereal weevil Sitophilus oryzae, bacteriomes are present as a single organ at the larval foregut-midgut junction, and in adults, at the apex of midgut mesenteric caeca and at the apex of the four ovarioles. While the adult midgut endosymbionts experience a drastic proliferation during early adulthood followed by complete elimination through apoptosis and autophagy, ovarian endosymbionts are maintained throughout the weevil lifetime by unknown mechanisms. Bacteria present in ovarian bacteriomes are thought to be involved in the maternal transmission of endosymbionts through infection of the female germline, but the exact mode of transmission is not fully understood. Here, we show that endosymbionts are able to colonize the germarium in one-week-old females, pinpointing a potential infection route of oocytes. To identify potential immune regulators of ovarian endosymbionts, we have analyzed the transcriptomes of the ovarian bacteriomes through young adult development, from one-day-old adults to sexually mature ones. In contrast with midgut bacteriomes, immune effectors are downregulated in ovarian bacteriomes at the onset of sexual maturation. We hypothesize that relaxation of endosymbiont control by antimicrobial peptides might allow bacterial migration and potential oocyte infection, ensuring endosymbiont transmission.

Coordination of host and endosymbiont gene expression governs endosymbiont growth and elimination in the cereal weevil Sitophilus spp.

BACKGROUND: Insects living in nutritionally poor environments often establish long-term relationships with intracellular bacteria that supplement their diets and improve their adaptive and invasive powers. Even though these symbiotic associations have been extensively studied on physiological, ecological, and evolutionary levels, few studies have focused on the molecular dialogue between host and endosymbionts to identify genes and pathways involved in endosymbiosis control and dynamics throughout host development. RESULTS: We simultaneously analyzed host and endosymbiont gene expression during the life cycle of the cereal weevil Sitophilus oryzae, from larval stages to adults, with a particular emphasis on emerging adults where the endosymbiont Sodalis pierantonius experiences a contrasted growth-climax-elimination dynamics. We unraveled a constant arms race in which different biological functions are intertwined and coregulated across both partners. These include immunity, metabolism, metal control, apoptosis, and bacterial stress response. CONCLUSIONS: The study of these tightly regulated functions, which are at the center of symbiotic regulations, provides evidence on how hosts and bacteria finely tune their gene expression and respond to different physiological challenges constrained by insect development in a nutritionally limited ecological niche. Video Abstract.

Efficient compartmentalization in insect bacteriomes protects symbiotic bacteria from host immune system.

BACKGROUND: Many insects house symbiotic intracellular bacteria (endosymbionts) that provide them with essential nutrients, thus promoting the usage of nutrient-poor habitats. Endosymbiont seclusion within host specialized cells, called bacteriocytes, often organized in a dedicated organ, the bacteriome, is crucial in protecting them from host immune defenses while avoiding chronic host immune activation. Previous evidence obtained in the cereal weevil Sitophilus oryzae has shown that bacteriome immunity is activated against invading pathogens, suggesting endosymbionts might be targeted and impacted by immune effectors during an immune challenge. To pinpoint any molecular determinants associated with such challenges, we conducted a dual transcriptomic analysis of S. oryzae's bacteriome subjected to immunogenic peptidoglycan fragments. RESULTS: We show that upon immune challenge, the bacteriome actively participates in the innate immune response via induction of antimicrobial peptides (AMPs). Surprisingly, endosymbionts do not undergo any transcriptomic changes, indicating that this potential threat goes unnoticed. Immunohistochemistry showed that TCT-induced AMPs are located outside the bacteriome, excluding direct contact with the endosymbionts. CONCLUSIONS: This work demonstrates that endosymbiont protection during an immune challenge is mainly achieved by efficient confinement within bacteriomes, which provides physical separation between host systemic response and endosymbionts. Video Abstract.

Phosphothioate oligodeoxynucleotides inhibit Plasmodium sporozoite gliding motility.

Plasmodium sporozoites, transmitted to the mammalian host through a mosquito bite, travel to the liver, where they invade hepatocytes, and develop into a form that is then able to infect red blood cells. In spite of the importance of innate immunity in controlling microbial infections, almost nothing is known about its role during the liver stage of a malaria infection. Here, we tested whether synthetic CpG phosphothioate (PS) oligodeoxynucleotides (ODNs), which bind to Toll-like receptor 9 (Tlr9), could have a protective effect on Plasmodium berghei infection in hepatocytes. Surprisingly, CpG PS-ODNs potently impair P. berghei infection in hepatoma cell lines independently of Tlr9 activation. Indeed, not only CpG but also non-CpG PS-ODNs, which do not activate Tlr9, decreased parasite infection. Moreover, the ability of PS-ODNs to impair infection was not due to an effect on the host but rather on the parasite itself. In fact, CpG PS-ODNs, as well as non-CpG PS-ODNs, impair parasite gliding motility. Furthermore, our analysis reveals that PS-ODNs inhibit parasite migration and invasion due to their negative charge, whereas development inside hepatocytes is undisturbed. Altogether, PS-ODNs might represent a new class of prophylactic anti-malaria agents, which hamper hepatocyte entry by Plasmodium sporozoites.

Endosymbiosis morphological reorganization during metamorphosis diverges in weevils.

Virtually all animals associate with beneficial symbiotic bacteria. Whether and how these associations are modulated across a host's lifecycle is an important question in disentangling animal-bacteria interactions. We recently reported a case of complete morphological reorganization of symbiosis during metamorphosis of the cereal weevil, Sitophilus oryzae. In this model, the bacteriome, a specialized organ that houses the intracellular bacterium Sodalis pierantonius, undergoes a two-phase remodeling program synchronously driven by host and endosymbiont, resulting in a localization shift and the formation of multiple new bacteriomes. Here, we provide comparative data in a closely-related coleopteran, the red palm weevil Rhynchophorus ferrugineus, which is associated with the ancestral endosymbiont Nardonella. Using cell imaging experiments, we show that the red pal weevil bacteriome remains unchanged during metamorphosis, hence contrasting with what we reported in the cereal weevil S. oryzae. These findings highlight the complexity and divergence of host-symbiont interactions and their intertwining with host development, even in closely-related species. Abbreviations: DAPI: 4',6-diamidino-2-phenylindole; FISH: Fluorescence in situ hybridization; T3SS: Type III secretion system.

Endosymbiosis as a source of immune innovation.

Some years ago, Lynn Margulis proposed to envision symbiosis as a source of evolutionary innovation. Here we revisit this concept in the context of insect nutritional endosymbiosis, and discuss recent data suggesting that host-endosymbiont coevolution has led to the selection of innovative strategies towards endosymbiont maintenance and control by the host immune system.

An IMD-like pathway mediates both endosymbiont control and host immunity in the cereal weevil Sitophilus spp.

Many insects developing on nutritionally unbalanced diets have evolved symbiotic associations with vertically transmitted intracellular bacteria (endosymbionts) that provide them with metabolic components, thereby improving the host's abilities to thrive on such poor ecological niches. While host-endosymbiont coevolutionary constraints are known to entail massive genomic changes in the microbial partner, host's genomic evolution remains elusive, particularly with regard to the immune system. In the cereal weevil Sitophilus spp., which houses Sodalis pierantonius, endosymbionts are secluded in specialized host cells, the bacteriocytes that group together as an organ, the bacteriome. We previously reported that at standard conditions, the bacteriome highly expresses the coleoptericin A (colA) antimicrobial peptide (AMP), which was shown to prevent endosymbiont escape from the bacteriocytes. However, following the insect systemic infection by pathogens, the bacteriome upregulates a cocktail of AMP encoding genes, including colA. The regulations that allow these contrasted immune responses remain unknown. In this short report, we provide evidence that an IMD-like pathway is conserved in two sibling species of cereal weevils, Sitophilus oryzae and Sitophilus zeamais. RNA interference (RNAi) experiments showed that imd and relish genes are essential for (i) colA expression in the bacteriome under standard conditions, (ii) AMP up-regulation in the bacteriome following a systemic immune challenge, and (iii) AMP systemic induction following an immune challenge. Histological analyses also showed that relish inhibition by RNAi resulted in endosymbiont escape from the bacteriome, strengthening the involvement of an IMD-like pathway in endosymbiont control. We conclude that Sitophilus' IMD-like pathway mediates both the bacteriome immune program involved in endosymbiont seclusion within the bacteriocytes and the systemic and local immune responses to exogenous challenges. This work provides a striking example of how a conserved immune pathway, initially described as essential in pathogen clearance, also functions in the control of mutualistic associations.

Effects of symbiotic status on cellular immunity dynamics in Sitophilus oryzae.

Many insects maintain intracellular symbiosis with mutualistic bacteria that improve their adaptive capabilities in nutritionally poor habitats. Adaptation of insect immune systems to such associations has been shown in several symbiotic consortia, including that of the rice weevil Sitophilus oryzae with the gammaproteobacterium Sodalis pierantonius. Although authors have mostly focused on the role of humoral immunity in host-symbiont interactions, recent studies suggest that symbiotic bacteria may also interfere with the cellular, hemocyte-based, immunity. Here, we have explored hemocyte dynamics in S. oryzae in the presence or absence of S. pierantonius, and in response to bacterial challenges. We have identified five morphotypes within larval hemocytes, whose abundance and morphometry drastically change along insect development. We show that hemocytes make part of the weevil immune system by responding to pathogenic infections. In contrast with previous results on other insect species, however, our analyses did not reveal any symbiotic-dependent modulation of the hemocyte global population.

Antimicrobial peptides and cell processes tracking endosymbiont dynamics.

Many insects sustain long-term relationships with intracellular symbiotic bacteria that provide them with essential nutrients. Such endosymbiotic relationships likely emerged from ancestral infections of the host by free-living bacteria, the genomes of which experience drastic gene losses and rearrangements during the host-symbiont coevolution. While it is well documented that endosymbiont genome shrinkage results in the loss of bacterial virulence genes, whether and how the host immune system evolves towards the tolerance and control of bacterial partners remains elusive. Remarkably, many insects rely on a 'compartmentalization strategy' that consists in secluding endosymbionts within specialized host cells, the bacteriocytes, thus preventing direct symbiont contact with the host systemic immune system. In this review, we compile recent advances in the understanding of the bacteriocyte immune and cellular regulators involved in endosymbiont maintenance and control. We focus on the cereal weevils Sitophilus spp., in which bacteriocytes form bacteriome organs that strikingly evolve in structure and number according to insect development and physiological needs. We discuss how weevils track endosymbiont dynamics through at least two mechanisms: (i) a bacteriome local antimicrobial peptide synthesis that regulates endosymbiont cell cytokinesis and helps to maintain a homeostatic state within bacteriocytes and (ii) some cellular processes such as apoptosis and autophagy which adjust endosymbiont load to the host developmental requirements, hence ensuring a fine-tuned integration of symbiosis costs and benefits.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Systemic infection generates a local-like immune response of the bacteriome organ in insect symbiosis.

Endosymbiosis is common in insects thriving in nutritionally unbalanced habitats. The cereal weevil, Sitophilus oryzae, houses Sodalis pierantonius, a Gram-negative intracellular symbiotic bacterium (endosymbiont), within a dedicated organ called a bacteriome. Recent data have shown that the bacteriome expresses certain immune genes that result in local symbiont tolerance and control. Here, we address the question of whether and how the bacteriome responds to insect infections involving exogenous bacteria. We have established an infection model by challenging weevil larvae with the Gram-negative bacterium Dickeya dadantii. We showed that D. dadantii infects host tissues and triggers a systemic immune response. Gene transcript analysis indicated that the bacteriome is also immune responsive, but it expresses immune effector genes to a lesser extent than the systemic and intestinal responses. Most genes putatively involved in immune pathways remain weakly expressed in the bacteriome following D. dadantii infection. Moreover, quantitative PCR experiments showed that the endosymbiont load is not affected by insect infection or the resulting bacteriome immune activation. Thus, the contained immune effector gene expression in the bacteriome may prevent potentially harmful effects of the immune response on endosymbionts, whilst efficiently protecting them from bacterial intruders.

Insects recycle endosymbionts when the benefit is over.

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.

Functional analysis of PGRP-LA in Drosophila immunity.

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.