Most animals have beneficial bacteria. Many of these associations are obligate: both partners are required. This project will establish whether the obligate symbiosis between an insect, the pea aphid, and the bacterium Buchnera is underpinned by shared metabolic pathways, meaning that both animal and bacteria contribute to the synthesis of nutrients needed by both partners. In these shared pathways, the nutrients are made using enzymes, some of which are coded by bacterial genes and others by animal genes, with intermediates being transferred between the animal and bacteria. This project will establish whether six essential amino acids are made by shared metabolic pathways in the pea aphid. The investigators will analyze the protein complement of the symbiosis, isolate candidate enzymes contributing to the shared pathways, determine their function, and quantify the movement of intermediates in the shared pathways by stable isotope labeling. The key results will be the identification of animal and bacterial enzymes that contribute to the shared metabolic pathways and the reactions they mediate, so that these shared metabolic pathways can be elucidated. This research will provide the first definitive evidence explaining why an animal-bacterial symbiosis is required by both partners. It will serve as a model to investigate the many other obligate symbioses in natural systems, and it will identify candidate targets for novel strategies to control aphid pests of crops. The project will contribute to the training of the next generation of scientists in insect science, proteomics and metabolic biochemistry. It will support the thesis research of a graduate student. Undergraduate summer students affiliated with this project will be recruited through an established Research Experience for Undergraduates program.
Many insects, including medical and agricultural pests, possess symbiotic bacteria on which they depend for sustained growth and reproduction. Typically, these bacteria have very small genomes and reduced numbers of genes. Aphids, which include important pests, depend on their association with the bacterium Buchnera, which provides specific nutrients, essential amino acids, which are in short supply in the aphid diet of plant sap. Linked to its small genome size, Buchnera lacks the genes for several enzymes in pathways for essential amino acid biosynthesis. The purpose of this project was to test whether enzymes in the aphid host compensate for the missing Buchnera enzymes, so allowing essential amino acid synthesis to proceed. Such shared metabolic pathways, with the shuttling of intermediate metabolites between insect and bacterium, are unprecedented. Strong evidence for shared metabolic pathways was obtained for five of the ten essential amino acids synthesized by Buchnera, based on an integrated analysis of the abundance of proteins (including enzymes) in the aphid and Buchnera, and analysis of the uptake and metabolic transformations of essential amino acid precursors by aphids and isolated Buchnera. Our experiments were guided by metabolic modeling based on the sequenced genomes of the aphid and bacterium. Buchnera has also been invoked to recycle aphid nitrogenous waste, specifically transforming ammonia into essential amino acids. Our studies refute this long-standing hypothesis, but demonstrate that aphid cells assimilate ammonia into carbon skeletons that only the Buchnera can synthesize, i.e. nitrogen recycling is mediated by the animal, not the bacterial partner. Shared metabolic pathways and animal-mediated nitrogen recycling, although never considered previously, are unlikely to be unique to the aphid-Buchnera symbiosis. These nutritional interactions may be widely distributed among the many microbial associations in insects and other animals. Aphids are major agricultural pests world-wide. There is an urgent need to develop novel pest control strategies linked to their increasing resistance to traditional insecticides. The identification of key genes whose function mediates aphid dependence on Buchnera bacteria lays the foundation for future research to target these genes. This offers a novel and highly specific strategy to disrupt the aphid-Buchnera interactions, and thereby abolish aphid growth and reproduction. This project has enabled in-depth research training in cutting-edge genomic, proteomic and metabolic analyses for two postdoctoral researchers, one graduate student and three undergraduate students. The expertise was disseminated more broadly by workshops linked to the techniques and concepts developed in this project (Metabolite Analysis, Insect Nutrition), with attendance by graduate students and postdoctoral researchers at Cornell University and other institutions in the region. The research has been communicated to the wider public through annual Department of Entomology open days (Insectapalooza!) and to schools via discussions and hands-on sessions with teachers (through Cornell Institute for Biology Teachers) and local schools, including the Birds, Bugs and Books Literacy Program. Addressing the complex issues of recruitment and retention of women in the life sciences, a two-day meeting Frontiers in the Life Sciences (with PI Douglas as co-organizer) was organized, where eight prestigious female scientists presented their research and provided small group mentoring for students and early-stage researchers in their specific disciplines.
