Functional Genomics of Buchnera and the Ecology of Aphid Hosts

Molecular Ecology (2006) 15, 1251–1261 doi: 10.1111/j.1365-294X.2005.02744.x FunctionalBlackwell Publishing, Ltd. genomics of Buchnera and the ecology of aphid hosts NANCY A. MORAN and PATRICK H. DEGNAN Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA Abstract In many animal groups, mutualistic bacterial symbionts play a central role in host ecology, by provisioning rare nutrients and thus enabling specialization on restricted diets. Among such symbionts, genomic studies are most advanced for Buchnera, the obligate symbiont of aphids, which feed on phloem sap. The contents of the highly reduced Buchnera genomes have verified its role in aphid nutrition. Comparisons of Buchnera gene sets indicate ongoing, irreversible gene losses that are expected to affect aphid nutritional needs. Furthermore, almost all regulatory genes have been eliminated, raising the question of whether and how gene expression responds to environmental change. Microarray studies on genome-wide expression indicate that Buchnera has evolved some constitutive changes in gene expression: homologues of heat stress genes have elevated transcript levels in Buchnera (relative to other bacteria) even in the absence of stress. Additionally, the microarray results indicate that responses to heat stress and to amino acid availability are both few and modest. Observed responses are consistent with control by the few ancestral regulators retained in the genome. Initial studies on the role of host genes in mediating the symbiosis reveal distinctive expression patterns in host cells harbouring Buchnera. In the near future, a complete genome of pea aphid will accelerate progress in understanding the functional integration of aphid and Buchnera genomes. Although information for other insect symbioses is relatively limited, studies on symbionts of carpenter ants and tsetse flies indicate many similarities to Buchnera. Keywords: bacteria, bacteriocyte, insects, nutrition, phloem-feeding, symbiosis Received 7 June 2005; revision accepted 10 August 2005 symbionts, especially for insect symbioses (e.g. Douglas Historical background & Prosser 1992; Sasaki & Ishikawa 1995; Febvay et al. 1999). Many invertebrates possess endosymbiotic bacteria that The use of molecular phylogenetic data has given robust are vertically transmitted from mother to progeny and confirmation to Buchner’s general view that associations that live in specialized organs. In his overview of animal between insects and their primary symbionts extend into symbioses, Buchner (1965) referred to these as ‘primary’ the deep evolutionary past, dating to the origins of major symbionts and proposed that their main function was groups such as aphids, psyllids, leafhoppers, carpenter ants, to provision nutrients that were limiting in the diet of their and others. Repeatedly, such studies have revealed con- hosts. In insects, such nutritional limitations are expected gruence between phylogenies of insect host and primary for a number of groups that have evolved specialized feed- symbiont, corroborating that symbionts were important ing habits, such as use of plant sap or vertebrate blood as players in the diversification of insect groups that are the sole food. A variety of experimental studies, involving dominant elements in modern terrestrial ecosystems. Thus, removal of native symbionts and/or manipulation of symbiosis was a prerequisite for the colonization of many diets have provided support for nutrient provisioning by specialized ecological niches and for the evolution of groups such as the sap-feeding and blood-feeding insects. In recent years, the emergence of genomics has expanded Correspondence: Nancy A. Moran, Fax: 520-621-3709; our understanding of symbiotic associations of insects and E-mail: [email protected] bacteria. Because bacterial genomes are compact and rich © 2006 Blackwell Publishing Ltd 1252 N. A. MORAN and P . H . DEGNAN in information regarding metabolic capabilities, the sequen- selective retention of particular biosynthetic genes demon- cing of complete genomes is relatively cost-effective. By strated a striking complementarity between host and providing a complete catalogue of genes and pathways symbiont metabolic capabilities. present in organisms, the sequences themselves give a direct Bacterial genomes are tightly packed with genes, indi- means of assessing functional contributions of symbionts cating that superfluous DNA is rapidly eliminated during to hosts. Genome sequences provide the foundation for the evolution of bacterial lineages (Mira et al. 2001; Lerat et al. analyses of the transcriptome or proteome and provide 2005). In symbiont genomes (and those of small genome the potential for assessing how symbiont functions are pathogens), the loss of DNA is extreme, implying strongly regulated in the context of the host environment. that genes that are retained with intact reading frames are The focus of this review is the best-studied insect– functionally significant to the organism. Other indicators symbiont system, that of Buchnera aphidicola and its are consistent with this inference: for example, different aphid hosts. Three genome sequences and several studies genes of a particular biosynthetic pathway are typically all of genome-wide gene expression have been published. We lost or all retained, even when the genes are positioned in summarize these results and relate them to some earlier, different parts of the chromosome. pre-genomics observations on aphid feeding and ecology. Remarkably, the Buchnera gene set is a subset of that of Together, these results suggest a role of Buchnera in both E. coli and closely related bacteria. This fact suggests that, the expansion of aphid ranges and in constraints on their at least in the case of Buchnera, becoming symbiotic did not geographical and host-plant distributions. Additionally, depend on acquiring novel genes. However, the Buchnera we compare results from Buchnera genomes to those from genome is highly derived and highly reduced, making it two other ancient bacteriocyte-associated insect symbionts impossible to determine whether some genes enabling for which genome sequences are available. host invasion were involved in early stages of the symbiosis. Subsequent co-adaptation with the host genome may have rendered symbiont invasion genes obsolete. Some symbionts Gene content and genome sizes of symbionts showing younger associations with insects, exemplified The first studies of symbiont gene content related to by Sodalis of tsetse flies, primary symbionts of Sitophilus function in insect hosts were those of Baumann and weevils, and secondary symbionts of aphids, possess Type coworkers, who cloned and sequenced fragments of the III Secretion Systems (Dale et al. 2001, 2002, 2005; Moran Buchnera chromosome from the aphid host Schizaphis et al. in press), similar to those used by pathogenic bacteria graminum (Baumann et al. 1995). They documented the to invade host cells. The underlying genes are known to presence of pathways for several essential amino acids, be horizontally transferred in many cases, and their acqui- confirming and extending previous evidence for a role of sition could be a step in the original evolution of an intra- Buchnera in provisioning hosts with these nutrients. Most cellular lifestyle. In Buchnera and other specialized symbionts, aphids feed exclusively on phloem sap, which contains early utilization of such a system might be obscured by limited amounts of essential amino acids; that is, the amino subsequent evolution of host mechanisms for ensuring acids used in proteins that animals cannot synthesize infection of bacteriocytes (the cells housing the symbionts) themselves. These pathways are very well studied in and by ongoing reduction of the symbiont genome. bacteria, particularly in the model system of Escherichia coli. The extent to which genome contents of insect symbionts The close relationship between Buchnera and E. coli, and the can be extended to infer metabolic capabilities is illustrated clearly defined homology of their genes, was a major by the detailed analysis of Zientz et al. (2004), which advantage in interpreting the new sequence data from elucidates the metabolic implications of the genomes of Buchnera. Buchnera, Blochmannia and Wigglesworthia. By the time the first Buchnera genome was published for the pea aphid Acyrthosiphon pisum (Shigenobu et al. 2000), Polyploidy it was known that Buchnera possessed genes for biosyn- thesis of most essential amino acids and that several of Many symbionts, including Buchnera, have very large these genes (for synthesis of tryptophan, isoleucine, valine, cell size: for example, the roughly spherical Buchnera cells leucine, and histidine) were transcribed (Baumann et al. 1995, are 3 µ in diameter and thus about 15× the volume of 1999). Complete genome sequences are the only means of an E. coli cell. In Buchnera at least, this increase in volume confidently showing what genes are absent from a genome, is accompanied by amplification of the genome. In and the first Buchnera genome sequence was perhaps most Acyrthosiphon pisum, individual Buchnera cells contain from notable for revealing which genes had been eliminated. 50 to over 100 chromosome copies, with the degree of These included most of those required for the synthesis of amplification varying with life cycle stage (Komaki & the nonessential amino acids, i.e. the amino acids that Ishikawa 1999, 2000). The functional significance of the

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