Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria

The interactions between herbivorous insects and their symbiotic micro-organisms can be influenced by the plant species on which the insects are reared, but the underlying mechanisms are not understood. Here, we identify plant nutrients, specifically amino acids, as a candidate factor affecting the impact of symbiotic bacteria on the performance of the phloem-feeding aphid Aphis fabae. Aphis fabae grew more slowly on the labiate plant Lamium purpureum than on an alternative host plant Vicia faba, and the negative effect of L. purpureum on aphid growth was consistently exacerbated by the bacterial secondary symbionts Regiella insecticola and Hamiltonella defensa, which attained high densities in L. purpureum-reared aphids. The amino acid content of the phloem sap of L. purpureum was very low; and A. fabae on chemically defined diets of low amino acid content also grew slowly and had elevated secondary symbiont densities. It is suggested that the phloem nutrient profile of L. purpureum promotes deleterious traits in the secondary symbionts and disturbs insect controls over bacterial abundance.     

Sources of variation in dietary requirements in an obligate nutritional symbiosis

The nutritional symbiosis between aphids and their obligate symbiont, Buchnera aphidicola, is often characterized as a highly functional partnership in which the symbiont provides the host with essential nutrients. Despite this, some aphid lineages exhibit dietary requirements for nutrients typically synthesized by Buchnera, suggesting that some aspect of the symbiosis is disrupted. To examine this phenomenon in the pea aphid, Acyrthosiphon pisum, populations were assayed using defined artificial diet to determine dietary requirements for essential amino acids (EAAs). Six clones exhibiting dependence on EAAs in their diet were investigated further. In one aphid clone, a mutation in a Buchnera amino acid biosynthesis gene could account for the clone''s requirement for dietary arginine. Analysis of aphid F₁ hybrids allowed separation of effects of the host and symbiont genomes, and revealed that both affect the requirement for dietary EAAs in the clones tested. Amino acid requirements were minimally affected by secondary symbiont infection. Our results indicate that variation among pea aphids in dependence on dietary amino acids can result from Buchnera mutation as well as variation in the host genotype.     

How the insect immune system interacts with an obligate symbiotic bacterium

The animal immune system provides defence against microbial infection, and the evolution of certain animal–microbial symbioses is predicted to involve adaptive changes in the host immune system to accommodate the microbial partner. For example, the reduced humoral immune system in the pea aphid Acyrthosiphon pisum, including an apparently non-functional immune deficiency (IMD) signalling pathway and absence of peptidoglycan recognition proteins (PGRPs), has been suggested to be an adaptation for the symbiosis with the bacterium Buchnera aphidicola. To investigate this hypothesis, the interaction between Buchnera and non-host cells, specifically cultured Drosophila S2 cells, was investigated. Microarray analysis of the gene expression pattern in S2 cells indicated that Buchnera triggered an immune response, including upregulated expression of genes for antimicrobial peptides via the IMD pathway with the PGRP-LC as receptor. Buchnera cells were readily taken up by S2 cells, but were subsequently eliminated over 1–2 days. These data suggest that Buchnera induces in non-host cells a defensive immune response that is deficient in its host. They support the proposed contribution of the Buchnera symbiosis to the evolution of the apparently reduced immune function in the aphid host.     

Injection of essential amino acids substitutes for bacterial supply in aposymbiotic pea aphids (Acyrthosiphon pisum)

The symbiotic bacteria Buchnera contribute to the nutrition of pea aphids, Acyrthosiphon pisum, through the provision of essential amino acids which are lacking in the diet. However, chemically defined diets, containing nutritionally adequate amounts of essential amino acids, fail to rescue aposymbiotic aphids, in which the bacteria have been disrupted with antibiotics. In this study the injection of a mixture of essential amino acids into the haemocoel of aposymbiotic aphids was shown to alleviate, at least partially, the impact of symbiont loss. Specifically, the total amino acid content in the tissues of aposymbiotic aphids was reduced by approximately 40% to levels comparable with symbiotic insects, and there was a 1.7-fold increase in the number of embryos, suggesting that the availability of essential amino acids promotes aphid protein synthesis by rejuvenating the free amino acid pool of aposymbiotic aphids. In addition, a similar effect on the total amino acid content was observed when phenylalanine alone, but not glutamine, lysine or tryptophan, was injected into the haemocoel of aposymbiotic aphids, and there was also a significant increase in the number of embryos following injection of phenylalanine or tryptophan alone. The impact of amino acid injection on the embryo complement of aposymbiotic aphids was limited to an increase in the number of embryos, with no increase in basal embryo size. It is proposed that older embryos may rely on their own complement of symbiotic bacteria for essential amino acid provisioning. Taken together, the data highlight the importance of bacterial provisioning of essential amino acids, particularly the aromatic amino acids, in the intact symbiosis.     

Genetic and metabolic determinants of nutritional phenotype in an insect-bacterial symbiosis

The pervasive influence of resident microorganisms on the phenotype of their hosts is exemplified by the intracellular bacterium Buchnera aphidicola, which provides its aphid partner with essential amino acids (EAAs). We investigated variation in the dietary requirement for EAAs among four pea aphid (Acyrthosiphon pisum) clones. Buchnera-derived nitrogen contributed to the synthesis of all EAAs for which aphid clones required a dietary supply, and to none of the EAAs for which all four clones had no dietary requirement, suggesting that low total dietary nitrogen may select for reduced synthesis of certain EAAs in some aphid clones. The sequenced Buchnera genomes showed that the EAA nutritional phenotype (i.e. the profile of dietary EAAs required by the aphid) cannot be attributed to sequence variation of Buchnera genes coding EAA biosynthetic enzymes. Metabolic modelling by flux balance analysis demonstrated that EAA output from Buchnera can be determined precisely by the flux of host metabolic precursors to Buchnera. Specifically, the four EAA nutritional phenotypes could be reproduced by metabolic models with unique profiles of host inputs, dominated by variation in supply of aspartate, homocysteine and glutamate. This suggests that the nutritional phenotype of the symbiosis is determined principally by host metabolism and transporter genes that regulate nutrient supply to Buchnera. Intraspecific variation in the nutritional phenotype of symbioses is expected to mediate partitioning of plant resources among aphid genotypes, potentially promoting the genetic subdivision of aphid populations. In this way, microbial symbioses may play an important role in the evolutionary diversification of phytophagous insects.     

Facultative 'secondary' bacterial symbionts and the nutrition of the pea aphid, Acyrthosiphon pisum

Abstract.  Facultative 'secondary' bacterial symbionts influence various traits of aphids, including plant utilization patterns and resistance to parasitoids. The present study is designed to test the hypothesis that these multiple effects are underlain by symbiont-mediated changes to the aphid requirement for the dominant dietary nutrients, sucrose and amino acids. The performance of pea aphids ( Acyrthosiphon pisum ) on chemically defined diets of systematically altered sucrose and amino acid content varies among eight parthenogenetic clones, with a pattern that does not match the aphid complements of secondary symbionts, Hamiltonella defensa , Regiella insecticola and Serratia symbiotica . Aphid performance is reduced, increased and unaffected by elimination of S. symbiotica , R. insecticola and H. defensa , respectively, but with no significant effect on the range of diets on which aphids performed well. It is concluded that the impact of secondary symbionts on aphid traits is most unlikely to have a purely nutritional basis.     

Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

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.     

Impact of a parasitoid on the bacterial symbiosis of its aphid host

Embryo production in aphids is absolutely dependent on the function of symbiotic bacteria, mainly Buchnera, and the growth and development of koinobiont parasitoids in aphids requires the diversion of nutrients from aphid embryo production to the parasitoid. The implication that the bacterial symbiosis may be promoted in parasitized aphids to support the growing parasitoid was explored by analysis of the number and biomass of mycetocytes, and the aphid cells bearing Buchnera, in the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) parasitized by the wasp Aphidius ervi Haliday (Hymenoptera: Braconidae). Aphids hosting a young larval parasitoid bore more mycetocytes of greater total biomass, and embryos of lower biomass than unparasitized aphids. Furthermore, one of the three aphid clones tested, which limited teratocyte growth (giant cells of parasitoid origin having a trophic role), bore smaller mycetocytes and larger embryos, than one or both of the two aphid clones with greater susceptibility to the parasitoid. These data suggest that susceptibility of the aphid‐Buchnera symbiosis to parasitoid‐mediated manipulation may, directly or indirectly, contribute to aphid susceptibility to parasitoid exploitation.     

Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis

Regulation of microbial population density is a necessity in stable symbiotic interactions. In Wolbachia symbiosis, both bacterial and host genotypes are involved in density regulation, but environmental factors may also affect bacterial population density. Here, we studied the interaction between three strains of Wolbachia in two divergent homozygous lines of the wasp Leptopilina heterotoma at two different temperatures. Wolbachia density varied between the two host genotypes at only one temperature. Moreover, at this temperature, reciprocal-cross F1 insects displayed identical Wolbachia densities, which were intermediate between the densities in the two parental lines. While these findings confirm that the host genotype plays an important role in Wolbachia density, they also highlight its interaction with environmental conditions, making possible the evolution of local adaptations for the regulation of Wolbachia density.     

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.     

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila'', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila'' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.     

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

Plant-ants feed their host plant, but above all a fungal symbiont to recycle nitrogen

In ant-plant symbioses, plants provide symbiotic ants with food and specialized nesting cavities (called domatia). In many ant-plant symbioses, a fungal patch grows within each domatium. The symbiotic nature of the fungal association has been shown in the ant-plant Leonardoxa africana and its protective mutualist ant Petalomyrmex phylax. To decipher trophic fluxes among the three partners, food enriched in (13)C and (15)N was given to the ants and tracked in the different parts of the symbiosis up to 660 days later. The plant received a small, but significant, amount of nitrogen from the ants. However, the ants fed more intensively the fungus. The pattern of isotope enrichment in the system indicated an ant behaviour that functions specifically to feed the fungus. After 660 days, the introduced nitrogen was still present in the system and homogeneously distributed among ant, plant and fungal compartments, indicating efficient recycling within the symbiosis. Another experiment showed that the plant surface absorbed nutrients (in the form of simple molecules) whether or not it is coated by fungus. Our study provides arguments for a mutualistic status of the fungal associate and a framework for investigating the previously unsuspected complexity of food webs in ant-plant mutualisms.     

Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts

Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell–cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell–cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends.     

Complete Genome sequence of Burkholderia phymatum STM815T,a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815^T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).     

Cheaper is not always worse: strongly protective isolates of a defensive symbiont are less costly to the aphid host

Defences against parasites are typically associated with costs to the host that contribute to the maintenance of variation in resistance. This also applies to the defence provided by the facultative bacterial endosymbiont Hamiltonella defensa, which protects its aphid hosts against parasitoid wasps while imposing life-history costs. To investigate the cost–benefit relationship within protected hosts, we introduced multiple isolates of H. defensa to the same genetic backgrounds of black bean aphids, Aphis fabae, and we quantified the protection against their parasitoid Lysiphlebus fabarum as well as the costs to the host (reduced lifespan and reproduction) in the absence of parasitoids. Surprisingly, we observed the opposite of a trade-off. Strongly protective isolates of H. defensa reduced lifespan and lifetime reproduction of unparasitized aphids to a lesser extent than weakly protective isolates. This finding has important implications for the evolution of defensive symbiosis and highlights the need for a better understanding of how strain variation in protective symbionts is maintained.     

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.