Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth.

The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, we performed a large-scale, systematic nutritional screen based on larval growth in 40 different and precisely controlled nutritional environments. We combined these results with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. We first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae and parsed such difference down to individual amino acids, vitamins, other micronutrients, and trace metals. We found that Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. Our systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.

Evolutionary novelty in the apoptotic pathway of aphids.

Apoptosis, a conserved form of programmed cell death, shows interspecies differences that may reflect evolutionary diversification and adaptation, a notion that remains largely untested. Among insects, the most speciose animal group, the apoptotic pathway has only been fully characterized in Drosophila melanogaster, and apoptosis-related proteins have been studied in a few other dipteran and lepidopteran species. Here, we studied the apoptotic pathway in the aphid Acyrthosiphon pisum, an insect pest belonging to the Hemiptera, an earlier-diverging and distantly related order. We combined phylogenetic analyses and conserved domain identification to annotate the apoptotic pathway in A. pisum and found low caspase diversity and a large expansion of its inhibitory part, with 28 inhibitors of apoptosis (IAPs). We analyzed the spatiotemporal expression of a selected set of pea aphid IAPs and showed that they are differentially expressed in different life stages and tissues, suggesting functional diversification. Five IAPs are specifically induced in bacteriocytes, the specialized cells housing symbiotic bacteria, during their cell death. We demonstrated the antiapoptotic role of these five IAPs using heterologous expression in a tractable in vivo model, the Drosophila melanogaster developing eye. Interestingly, IAPs with the strongest antiapoptotic potential contain two BIR and two RING domains, a domain association that has not been observed in any other species. We finally analyzed all available aphid genomes and found that they all show large IAP expansion, with new combinations of protein domains, suggestive of evolutionarily novel aphid-specific functions.

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.

Aphid BCR4 Structure and Activity Uncover a New Defensin Peptide Superfamily.

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.

Bacteriocyte cell death in the pea aphid/Buchnera symbiotic system.

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.

The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

BACKGROUND: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species 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 of D. vitifoliae as 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.

Correction to: The genome sequence of the grape phylloxera provides insights into the evolution, adaptation, and invasion routes of an iconic pest.

An amendment to this paper has been published and can be accessed via the original article.

Identification of Plant Virus Receptor Candidates in the Stylets of Their Aphid Vectors.

Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a noncirculative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a noncirculative virus. A peptide motif present at the C termini of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus Furthermore, silencing the stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of noncirculative plant viruses.IMPORTANCE Most noncirculative plant viruses transmitted by insect vectors bind to their mouthparts. They are acquired and inoculated within seconds when insects hop from plant to plant. The receptors involved remain totally elusive due to a long-standing technical bottleneck in working with insect cuticle. Here we characterize the role of the two first cuticular proteins ever identified in arthropod mouthparts. A domain of these proteins is directly accessible at the surface of the cuticle of the acrostyle, an organ at the tip of aphid stylets. The acrostyle has been shown to bind a plant virus, and we consistently demonstrated that one of the identified proteins is involved in viral transmission. Our findings provide an approach to identify proteins in insect mouthparts and point at an unprecedented gene candidate for a plant virus receptor.

PacBio Hi-Fi genome assembly of Sipha maydis, a model for the study of multipartite mutualism in insects.

Dependence on multiple nutritional endosymbionts has evolved repeatedly in insects feeding on unbalanced diets. However, reference genomes for species hosting multi-symbiotic nutritional systems are lacking, even though they are essential for deciphering the processes governing cooperative life between insects and anatomically integrated symbionts. The cereal aphid Sipha maydis is a promising model for addressing these issues, as it has evolved a nutritional dependence on two bacterial endosymbionts that complement each other. In this study, we used PacBio High fidelity (HiFi) long-read sequencing to generate a highly contiguous genome assembly of S. maydis with a length of 410 Mb, 3,570 contigs with a contig N50 length of 187 kb, and BUSCO completeness of 95.5%. We identified 117 Mb of repetitive sequences, accounting for 29% of the genome assembly, and predicted 24,453 protein-coding genes, of which 2,541 were predicted enzymes included in an integrated metabolic network with the two aphid-associated endosymbionts. These resources provide valuable genetic and metabolic information for understanding the evolution and functioning of multi-symbiotic systems in insects.

Reference genes to study the sex-biased expression of genes regulating Drosophila metabolism.

Sex is an important variable in biology. Notable differences have been observed between male and female Drosophila in regulation of metabolism, in response to nutritional challenges, and in phenotypes relevant for obesity and metabolic disorders. The differences between males and females can be expected to result from differences in gene expression. We observed that expression levels of reference genes commonly used for normalization of qRT-PCR results such as GAPDH, ß-actin, and 18SrRNA, show prominent sexual dimorphism. Since this will impact relative expression and conclusions related to that, we performed a systematic analysis of candidate reference genes with the objective of identifying reference genes with stable expression in male and female Drosophila. These reference genes (LamCa, ßTub60D and ßTub97EF) were then used to assess sex-specific differences in expression of metabolism associated genes. Additionally, we evaluated the utility of these reference genes following a nutritional challenge and showed that LamCa and ßtub97EF are stably expressed between sexes and under different nutritional conditions and are thus suitable as reference genes. Our results highlight the importance of evaluating the stability of reference genes when sex-specific differences in gene expression are studied, and identify structural genes as a category worth exploring as reference genes in other species. Finally, we also uncovered hitherto unknown sexually dimorphic expression of a number of metabolism-associated genes, information of interest to others working in the field of metabolic disorders.

Proteogenomic reconstruction of organ-specific metabolic networks in an environmental sentinel species, the amphipod Gammarus fossarum.

Metabolic pathways are affected by the impacts of environmental contaminants underlying a large variability of toxic effects across different species. However, the systematic reconstruction of metabolic pathways remains limited in environmental sentinel species due to the lack of available genomic data in many taxa of animal diversity. In this study we used a multi-omics approach to reconstruct the most comprehensive map of metabolic pathways for a crustacean model in biomonitoring, the amphipod Gammarus fossarum in order to improve the knowledge of the metabolism of this sentinel species. We revisited the assembly of RNA-seq data by de novo approaches to reduce RNA contaminants and transcript redundancy. We also acquired extensive mass spectrometry shotgun proteomic data on several organs from a reference population of G. fossarum males and females to identify organ-specific metabolic profiles. The G. fossarum metabolic pathway reconstruction (available through the metabolic database GamfoCyc) was performed by adapting the genomic tool CycADS and we identified 377 pathways representing 7630 annotated enzymes, 2610 enzymatic reactions and the expression of 858 enzymes was experimentally validated by proteomics. To our knowledge, our analysis provides for the first time a systematic metabolic pathway reconstruction and the proteome profiles of these pathways at the organ level in this sentinel species. As an example, we show an elevated abundance in enzymes involved in ATP biosynthesis and fatty acid beta-oxidation indicative of the high-energy requirement of the gills, or the key anabolic and detoxification role of the hepatopancreatic caeca, as exemplified by the specific expression of the retinoid biosynthetic pathways and glutathione synthesis. In conclusion, the multi-omics data integration performed in this study provides new resources to investigate metabolic processes in crustacean amphipods and their role in mediating the effects of environmental contaminant exposures in sentinel species. SYNOPSIS: This study provide the first evidence that it is possible to combine multiple omics data to exhaustively describe the metabolic network of a model species in ecotoxicology, Gammarus fossarum, for which a reference genome is not yet available.

Genomic analysis of the regulatory elements and links with intrinsic DNA structural properties in the shrunken genome of Buchnera.

BACKGROUND: Buchnera aphidicola is an obligate symbiotic bacterium, associated with most of the aphididae, whose genome has drastically shrunk during intracellular evolution. Gene regulation in Buchnera has been a matter of controversy in recent years as the combination of genomic information with the experimental results has been contradictory, refuting or arguing in favour of a functional and responsive transcription regulation in Buchnera.The goal of this study was to describe the gene transcription regulation capabilities of Buchnera based on the inventory of cis- and trans-regulators encoded in the genomes of five strains from different aphids (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, Cinara cedri and Cinara tujafilina), as well as on the characterisation of some intrinsic structural properties of the DNA molecule in these bacteria. RESULTS: Interaction graph analysis shows that gene neighbourhoods are conserved between E. coli and Buchnera in structures called transcriptons, interactons and metabolons, indicating that selective pressures have acted on the evolution of transcriptional, protein-protein interaction and metabolic networks in Buchnera. The transcriptional regulatory network in Buchnera is composed of a few general DNA-topological regulators (Nucleoid Associated Proteins and topoisomerases), with the quasi-absence of any specific ones (except for multifunctional enzymes with a known gene expression regulatory role in Escherichia coli, such as AlaS, PepA and BolA, and the uncharacterized hypothetical regulators YchA and YrbA). The relative positioning of regulatory genes along the chromosome of Buchnera seems to have conserved its ancestral state, despite the genome erosion. Sigma-70 promoters with canonical thermodynamic sequence profiles were detected upstream of about 94% of the CDS of Buchnera in the different aphids. Based on Stress-Induced Duplex Destabilization (SIDD) measurements, unstable σ70 promoters were found specifically associated with the regulator and transporter genes. CONCLUSIONS: This genomic analysis provides supporting evidence of a selection of functional regulatory structures and it has enabled us to propose hypotheses concerning possible links between these regulatory elements and the DNA-topology (i.e., supercoiling, curvature, flexibility and base-pair stability) in the regulation of gene expression in the shrunken genome of Buchnera.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

BACKGROUND: Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. RESULTS: We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development. CONCLUSIONS: Our data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

Gene Self-Expressive Networks as a Generalization-Aware Tool to Model Gene Regulatory Networks.

Self-expressiveness is a mathematical property that aims at characterizing the relationship between instances in a dataset. This property has been applied widely and successfully in computer-vision tasks, time-series analysis, and to infer underlying network structures in domains including protein signaling interactions and social-networks activity. Nevertheless, despite its potential, self-expressiveness has not been explicitly used to infer gene networks. In this article, we present Generalizable Gene Self-Expressive Networks, a new, interpretable, and generalization-aware formalism to model gene networks, and we propose two methods: GXN•EN and GXN•OMP, based respectively on ElasticNet and OMP (Orthogonal Matching Pursuit), to infer and assess Generalizable Gene Self-Expressive Networks. We evaluate these methods on four Microarray datasets from the DREAM5 benchmark, using both internal and external metrics. The results obtained by both methods are comparable to those obtained by state-of-the-art tools, but are fast to train and exhibit high levels of sparsity, which make them easier to interpret. Moreover we applied these methods to three complex datasets containing RNA-seq informations from different mammalian tissues/cell-types. Lastly, we applied our methodology to compare a normal vs. a disease condition (Alzheimer), which allowed us to detect differential expression of genes' sub-networks between these two biological conditions. Globally, the gene networks obtained exhibit a sparse and modular structure, with inner communities of genes presenting statistically significant over/under-expression on specific cell types, as well as significant enrichment for some anatomical GO terms, suggesting that such communities may also drive important functional roles.

GReNaDIne: A Data-Driven Python Library to Infer Gene Regulatory Networks from Gene Expression Data.

Context: Inferring gene regulatory networks (GRN) from high-throughput gene expression data is a challenging task for which different strategies have been developed. Nevertheless, no ever-winning method exists, and each method has its advantages, intrinsic biases, and application domains. Thus, in order to analyze a dataset, users should be able to test different techniques and choose the most appropriate one. This step can be particularly difficult and time consuming, since most methods' implementations are made available independently, possibly in different programming languages. The implementation of an open-source library containing different inference methods within a common framework is expected to be a valuable toolkit for the systems biology community. Results: In this work, we introduce GReNaDIne (Gene Regulatory Network Data-driven Inference), a Python package that implements 18 machine learning data-driven gene regulatory network inference methods. It also includes eight generalist preprocessing techniques, suitable for both RNA-seq and microarray dataset analysis, as well as four normalization techniques dedicated to RNA-seq. In addition, this package implements the possibility to combine the results of different inference tools to form robust and efficient ensembles. This package has been successfully assessed under the DREAM5 challenge benchmark dataset. The open-source GReNaDIne Python package is made freely available in a dedicated GitLab repository, as well as in the official third-party software repository PyPI Python Package Index. The latest documentation on the GReNaDIne library is also available at Read the Docs, an open-source software documentation hosting platform. Contribution: The GReNaDIne tool represents a technological contribution to the field of systems biology. This package can be used to infer gene regulatory networks from high-throughput gene expression data using different algorithms within the same framework. In order to analyze their datasets, users can apply a battery of preprocessing and postprocessing tools and choose the most adapted inference method from the GReNaDIne library and even combine the output of different methods to obtain more robust results. The results format provided by GReNaDIne is compatible with well-known complementary refinement tools such as PYSCENIC.

Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.

Ants are among the most abundant terrestrial invertebrate predators on Earth. To overwhelm their prey, they employ several remarkable behavioral, physiological, and biochemical innovations, including an effective paralytic venom. Ant venoms are thus cocktails of toxins finely tuned to disrupt the physiological systems of insect prey. They have received little attention yet hold great promise for the discovery of novel insecticidal molecules. To identify insect-neurotoxins from ant venoms, we screened the paralytic activity on blowflies of nine synthetic peptides previously characterized in the venom of Tetramorium bicarinatum. We selected peptide U11, a 34-amino acid peptide, for further insecticidal, structural, and pharmacological experiments. Insecticidal assays revealed that U11 is one of the most paralytic peptides ever reported from ant venoms against blowflies and is also capable of paralyzing honeybees. An NMR spectroscopy of U11 uncovered a unique scaffold, featuring a compact triangular ring helix structure stabilized by a single disulfide bond. Pharmacological assays using Drosophila S2 cells demonstrated that U11 is not cytotoxic, but suggest that it may modulate potassium conductance, which structural data seem to corroborate and will be confirmed in a future extended pharmacological investigation. The results described in this paper demonstrate that ant venom is a promising reservoir for the discovery of neuroactive insecticidal peptides.

Multimodal dynamic response of the Buchnera aphidicola pLeu plasmid to variations in leucine demand of its host, the pea aphid Acyrthosiphon pisum.

Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the γ-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.

Bacteriocyte plasticity in pea aphids facing amino acid stress or starvation during development.

An important contributing factor to the evolutionary success of insects is nutritional association with microbial symbionts, which provide the host insects with nutrients lacking in their unbalanced diets. These symbionts are often compartmentalized in specialized cells of the host, the bacteriocytes. Even though bacteriocytes were first described more than a century ago, few studies have explored their dynamics throughout the insect life cycle and in response to environmental stressors. Here, we use the Buchnera aphidicola/pea aphid symbiotic system to study how bacteriocytes are regulated in response to nutritional stress throughout aphid development. Using artificial diets, we analyzed the effects of depletion or excess of phenylalanine or leucine, two amino acids essential for aphid growth and whose biosynthetic pathways are shared between the host and the symbiont. Bacteriocytes responded dynamically to those treatments, while other tissues showed no obvious morphological change. Amino acid depletion resulted in an increase in bacteriocyte numbers, with the extent of the increase depending on the amino acid, while excess either caused a decrease (for leucine) or an increase (for phenylalanine). Only a limited impact on survival and fecundity was observed, suggesting that the adjustment in bacteriocyte (and symbiont) numbers is sufficient to withstand these nutritional challenges. We also studied the impact of more extreme conditions by exposing aphids to a 24 h starvation period at the beginning of nymphal development. This led to a dramatic drop in aphid survival and fecundity and a significant developmental delay. Again, bacteriocytes responded dynamically, with a considerable decrease in number and size, correlated with a decrease in the number of symbionts, which were prematurely degraded by the lysosomal system. This study shows how bacteriocyte dynamics is integrated in the physiology of insects and highlights the high plasticity of these cells.

The Di-Symbiotic Systems in the Aphids Sipha maydis and Periphyllus lyropictus Provide a Contrasting Picture of Recent Co-Obligate Nutritional Endosymbiosis in Aphids.

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont Buchnera aphidicola is metabolically complemented by an additional nutritional symbiont acquired more recently. Deciphering how different symbionts integrate both metabolically and anatomically in such systems is crucial to understanding how complex nutritional symbiotic systems function and evolve. In this study, we sequenced and analyzed the genomes of the symbionts B. aphidicola and Serratia symbiotica associated with the Chaitophorinae aphids Sipha maydis and Periphyllus lyropictus. Our results show that, in these two species, B. aphidicola and S. symbiotica complement each other metabolically (and their hosts) for the biosynthesis of essential amino acids and vitamins, but with distinct metabolic reactions supported by each symbiont depending on the host species. Furthermore, the S. symbiotica symbiont associated with S. maydis appears to be strictly compartmentalized into the specialized host cells housing symbionts in aphids, the bacteriocytes, whereas the S. symbiotica symbiont associated with P. lyropictus exhibits a highly invasive phenotype, presumably because it is capable of expressing a larger set of virulence factors, including a complete flagellum for bacterial motility. Such contrasting levels of metabolic and anatomical integration for two S. symbiotica symbionts that were recently acquired as nutritional co-obligate partners reflect distinct coevolutionary processes specific to each association.