Drosophila melanogaster (adult male & larva)
Rarefaction curves of OTUs clustered at different %ID across lifestages of D. melanogaster Canton-S [Wong et al. 2011 Env Microbiol.13, 1889]
The symbiotic bacteria Buchnera are located in specialized cells called bacteriocytes (shown green in figure) in the aphid body cavity. [Drawing by Tomás Lazo]
FISH micrograph of sectioned black bean aphid Aphis fabae hybridized with FITC-labelled DNA probe specific to 16S rRNA of Buchnera (green), with the DNA stain DAPI (blue) as counterstain. [Micrograph by Simon Chandler]
Each Buchnera cell is separated from the bacteriocyte cytoplasm by three membranes, interpreted as the bacterial inner membrane, bacterial outer membrane and the outermost aphid symbiosomal membrane (arrow). All metabolites transferred between the host and symbiont must cross these three membranes. [Micrograph by Angela Douglas]
Buchnera cells are transmitted vertically from the maternal bacteriocytes to the blastoderm embryos in parthenogenetic aphids (shown) or to the cytoplasm of sexual eggs. [Micrograph by Tom Wilkinson]
Immunolocalization of GroEL protein of the bacterium Buchnera in sectioned pea aphid, using a monoclonal antibody we raised against recombinant Buchnera GroEL. GroEL (red) is detected in Buchnera localized in aphid bacteriocytes in the body cavity and embryos [Bouvaine et al. 2011. J. gen Virol 92: 1467 
Insect interactions with resident microorganisms
We live in a microbial world. Animals, including insects, are colonized by benign microorganisms. Many animals benefit from microbial-derived nutrients and microbial protection against pathogens and other natural enemies.
We study two types of association: gut symbioses, especially in Drosophila; and intracellular symbioses, especially in plant sap feeding insects.
1. The Gut Microbiota of Drosophila
The GI tract of Drosophila is chronically infected with bacteria. We are investigating how these bacteria influence the health and wellbeing of their insect host. Because microbial associations with the animal GI tract are ancient - probably as ancient as animals - these studies should reveal fundamental biology of how microorganisms influence human health, and how disruption of the host-microbial interactions is linked to chronic metabolic and immunological diseases.
We have conducted a comprehensive analysis of the microbiota in Drosophila melanogaster by 454 sequencing of 16S rRNA gene amplicons. Most life stages of Drosophila bear <30 taxa (97% sequence identity) and are dominated by just 5 bacterial species (Figure). This provides a superb opportunity to link bacterial function to individual species.
Figure: Percentage composition of bacterial species at different lifestages of D. melanogaster [Redrawn from data in Wong CN, Ng P and Douglas AE (2011) Low diversity bacterial community in the gut of the fruitfly Drosophila melanogaster. Environmental Microbiology 13: 1889-1900. Pubmed]
We are currently investigating the impact of the gut microbiota on Drosophila nutrition, using axenically reared insects and various dietary manipulations, with host gene expression, metabolism and performance as read-outs of the host-microbial interaction.
2. Intracellular Symbioses in Insects
especially aphids and other plant sap feeding hemipterans
Various insects possess beneficial microorganisms housed in specialized cells called bacteriocytes. There is no parallel to these relationships among mammals (in which all known intracellular microbes are pathogens).
The symbiotic bacteria provide nutrients to their insect hosts
The symbiotic bacteria Buchnera aphidicola provide their aphid
hosts with essential amino acids. These nutrients are in short supply
in the aphid diet of plant phloem sap. For example, the pea aphid
obtains 4 x its total nitrogen requirement from the phloem sap of its
host plant Vicia faba (fava bean), but the essential amino acid
content is very low. We have calculated that the sustained growth of
the aphid is dependent on the supply of all 10 essential amino acids
from the Buchnera bacteria.
Figure. Buchnera-derived essential amino acids supporting the growth of pea aphid larvae.[Gündüz and Douglas 2009. Proceedings of the Royal Society of London B 276, 987-991. Pubmed link]
The aphid bacteriocyte: How to be a host cell
We are investigating how the symbiosis is maintained in the aphid bacteriocyte. Our recent 
quantitative proteomics of the aphid bacteriocyte, in collaboration with Klaas van Wijk, has revealed
- Most proteins enriched in bacteriocytes are metabolic enzymes, especially those predicted from metabolic modeling (see below) to synthesize metabolites required by Buchnera
- All Buchnera enzymes in essential amino acid synthesis are expressed
- The complement of proteins in Buchnera cells is defined by the Buchnera genome, with no evidence for specific targeting of host proteins to the Buchnera cells.
Figure The proteome of the pea aphid. A, 1D-SDS-PAGE gel lanes of the five different types of fractions (WB = whole body,BC = bacteriocytes, Bu-1 = partially purified Buchnera, Bu-2 = Percoll-purified Buchnera, and BR= Buchnera-free bacteriocyte fraction) stained with Coomassie Brilliant Blue. B, C, Total number of proteins (B) and total mass of proteins (C) based on NadjSPC of aphid origin (gray) and Buchnera origin (white). D, Functional classification of quantified proteins, expressed as number of proteins (top row) and abundance, expressed as mean NSAF normalized to total aphid or Buchnera proteins (bottom row). [Figure from Poliakov A, Russell CW, Ponnala L, Hoops HJ, Sun Q, Douglas AE and van Wijk KJ, 2011. Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis. Molecular and Cellular Proteomics 10: M10.007039 Pubmed link]
The Buchnera metabolic network: how to be an intracellular symbiont
Linked to its small genome size (0.64 Mb), Buchnera has very limited metabolic capabilities. To investigate how its metabolism is integrated with the host, we have reconstructed the metabolic network of Buchnera APS from the pea aphid. The network is extremely fragile with >90% of the reactions required for viability in silico. These traits are suggested to make Buchnera intolerant of environmental variation.
Figure The metabolic network of Buchnera comprises 196 gene products, 240 metabolites and 263 reactions. Nearly 35% of the reactions are involved in essential amino acid synthesis. The metabolites consumed in the model are shown in red, by-products are yellow, and components of the biomass reaction, including essential amino acids (large symbols) are blue. [Thomas et al. 2009. BMC Systems Biology 3, e24 Pubmed Link]
We have investigated the distribution of metabolic flux through the Buchnera metabolic network by flux balance analysis. From this, we have identified the nutrients that the Buchnera requires for growth and essential amino acid production. The Figure shows these nutrients and their predicted flux, normalized to glucose = 100 (carbon sources red; non-essential amino acids blue; inorganic compounds green; others purple) [From MacDonald SJ, Thomas GH and Douglas AE, 2011. Genetic and metabolic determinants of nutritional phenotype in an insect-bacterial symbiosis. Molecular Ecology 20: 2073-84 Pubmed link]
In parallel, we have contributed to the annotated of the metabolism genes in the pea aphid by the International Aphid Genomics Consortium, revealing remarkable complementarity between the gene content of aphid and Buchnera. Specifically, Buchnera lacks key genes in the synthesis of 5 essential amino acids, and the aphid possesses these - but no other - genes in these pathways. We are currently testing the hypothesis that these pathways are coupled between the symbiotic partners, in collaboration with Dr George Jander.