Matching the supply of bacterial nutrients to the nutritional demand of the animal host.

Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.

Shared metabolic pathways in a coevolved insect-bacterial symbiosis.

The symbiotic bacterium Buchnera aphidicola lacks key genes in the biosynthesis of five essential amino acids (EAAs), and yet its animal hosts (aphids) depend on the symbiosis for the synthesis of these EAAs (isoleucine, leucine, methionine, phenylalanine, and valine). We tested the hypothesis, derived from genome annotation, that the missing Buchnera reactions are mediated by host enzymes, with the exchange of metabolic intermediates between the partners. The specialized host cells bearing Buchnera were separated into a Buchnera fraction and a Buchnera-free host cell fraction (HF). Addition of HF to isolated Buchnera preparations significantly increased the production of leucine and phenylalanine, and recombinant enzymes mediating the final reactions in branched-chain amino acid and phenylalanine synthesis rescued the production of these EAAs by Buchnera preparations without HF. The likely precursors for the missing proximal reactions in isoleucine and methionine synthesis were identified, and they differed from predictions based on genome annotations: synthesis of 2-oxobutanoate, the aphid-derived precursor of isoleucine synthesis, was stimulated by homoserine and not threonine via threonine dehydratase, and production of the homocysteine precursor of methionine was driven by cystathionine, not cysteine, via reversal of the transsulfuration pathway. The evolution of shared metabolic pathways in this symbiosis can be attributed to host compensation for genomic deterioration in the symbiont, involving changes in host gene expression networks to recruit specific enzymes to the host cell.

The central role of the host cell in symbiotic nitrogen metabolism.

Symbiotic nitrogen recycling enables animals to thrive on nitrogen-poor diets and environments. It traditionally refers to the utilization of animal waste nitrogen by symbiotic micro-organisms to synthesize essential amino acids (EAAs), which are translocated back to the animal host. We applied metabolic modelling and complementary metabolite profiling to investigate nitrogen recycling in the symbiosis between the pea aphid and the intracellular bacterium Buchnera, which synthesizes EAAs. The results differ from traditional notions of nitrogen recycling in two important respects. First, aphid waste ammonia is recycled predominantly by the host cell (bacteriocyte) and not Buchnera. Host cell recycling is mediated by shared biosynthetic pathways for four EAAs, in which aphid transaminases incorporate ammonia-derived nitrogen into carbon skeletons synthesized by Buchnera to generate EAAs. Second, the ammonia substrate for nitrogen recycling is derived from bacteriocyte metabolism, such that the symbiosis is not a sink for nitrogenous waste from other aphid organs. Host cell-mediated nitrogen recycling may be general among insect symbioses with shared EAA biosynthetic pathways generated by the loss of symbiont genes mediating terminal reactions in EAA synthesis.

Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis.

Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.

Interactions between imidacloprid and Metarhizium brunneum on adult Asian longhorned beetles (Anoplophora glabripennis (Motschulsky)) (Coleoptera: Cerambycidae).

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), a longhorned beetle species native to Asia, has been introduced into several North American and European cities. Currently eradication and preventive measures are limited to identifying and destroying infested trees and protecting uninfested trees with trunk or soil-injections of the systemic insecticide imidacloprid. Because entomopathogenic fungi like Metarhizium brunneum Petch have been identified as virulent against these beetles we conducted several tests to determine the compatibility of the two agents in combination. Radial hyphal growth and the sporulation capacity of M. brunneum on Sabouraud dextrose agar with yeast were not significantly affected by the presence of imidacloprid. In a 2×3 factorial experiment investigating interactions between exposure to imidacloprid and M. brunneum we observed no effect of imidacloprid alone on beetle survival when beetles were given a single dose of 10 or 100 ppm compared to control insects. We observed a significant effect of exposure to M. brunneum, and a significant interaction between imidacloprid and M. brunneum representing a synergistic effect of dual treatment. Beetles exposed to the fungus alone lived significantly longer compared to insects treated with a single dose of 100 ppm imidacloprid (9.5 vs. 6.5d). Consumption of striped maple twigs by beetles exposed to imidacloprid, across concentrations, was reduced 48% compared to control insects, where as consumption by M. brunneum-exposed beetles was reduced by 16% over the first 6-days of the test period. Beetles fed 100 ppm imidacloprid consumed 32% less over the first 3d compared to beetles not exposed to imidacloprid and thereafter consumed as much as beetles not fed 100 ppm imidacloprid. M. brunneum-exposed beetles consumed significantly less food than control insects throughout the test period, and beetles treated with imidacloprid produced significantly fewer conidia compared to beetles not treated with imidacloprid.