The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched‐chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

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 pomorum^WJL (Ap^WJL) and Lactobacillus plantarum^NC8 (Lp^NC8), 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 Ap^WJL and Lp^NC8 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.

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. Significance Beneficial symbiotic associations, ubiquitously found in nature, have led to the emergence of eukaryotic cells, the bacteriocytes, specialized in harboring microbial partners. One of the most fundamental questions concerning these enigmatic cells is how organismal homeostasis controls their elimination. Here we report that aphid bacteriocytes have evolved a form of cell death distinct from the conserved cell-death mechanisms hitherto characterized. This cell-death mechanism is a nonapoptotic multistep process that starts with the hypervacuolation of the endoplasmic reticulum, followed by a cascade of cellular stress responses. Our findings provide a framework to study biological functioning of bacteriocytes and the cellular mechanisms associated with symbiosis and contribute to the understanding of eukaryotic cell-death diversity.

RNA interference (RNAi) has been widely and successfully used for gene inactivation in insects, including aphids, where dsRNA administration can be performed either by feeding or microinjection. However, several aspects related to the aphid response to RNAi, as well as the influence of the administration method on tissue response, or the mixed success to observe phenotypes specific to the gene targeted, are still unclear in this insect group. In the present study, we made the first direct comparison of two administration methods (injection or feeding) for delivery of dsRNA targeting the cathepsin-L gene in the pea aphid, Acyrthosiphon pisum. In order to maximize the possibility of discovering specific phenotypes, the effect of the treatment was analyzed in single individual aphids at the level of five body compartments: the bacteriocytes, the gut, the embryonic chains, the head and the remaining body carcass. Our analysis revealed that gene expression knockdown effect in each single body compartment was dependent on the administration method used, and allowed us to discover new functions for the cathepsin-L gene in aphids. Injection of cathepsin-L dsRNA was much more effective on carcass and head, inducing body morphology alterations, and suggesting a novel role of this gene in the molting of these insects. Administration by feeding provoked cathepsin-L knockdown in the gut and specific gut epithelial cell alteration, therefore allowing a better characterization of tissue specific role of this gene in aphids.

Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, these results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development.

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.