Co-option of a conserved host glutamine transporter facilitates aphid/Buchnera metabolic integration.

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

SymbiQuant: A Machine Learning Object Detection Tool for Polyploid Independent Estimates of Endosymbiont Population Size.

Quantifying the size of endosymbiont populations is challenging because endosymbionts are typically difficult or impossible to culture and commonly polyploid. Current approaches to estimating endosymbiont population sizes include quantitative PCR (qPCR) targeting endosymbiont genomic DNA and flow-cytometry. While qPCR captures genome copy number data, it does not capture the number of bacterial cells in polyploid endosymbiont populations. In contrast, flow cytometry can capture accurate estimates of whole host-level endosymbiont population size, but it is not readily able to capture data at the level of endosymbiotic host cells. To complement these existing approaches for estimating endosymbiont population size, we designed and implemented an object detection/segmentation tool for counting the number of endosymbiont cells in micrographs of host tissues. The tool, called SymbiQuant, which makes use of recent advances in deep neural networks includes a graphic user interface that allows for human curation of tool output. We trained SymbiQuant for use in the model aphid/Buchnera endosymbiosis and studied Buchnera population dynamics and phenotype over aphid postembryonic development. We show that SymbiQuant returns accurate counts of endosymbionts, and readily captures Buchnera phenotype. By replacing our training data with data composed of annotated microscopy images from other models of endosymbiosis, SymbiQuant has the potential for broad application. Our tool, which is available on GitHub, adds to the repertoire of methods researchers can use to study endosymbiosis at the organismal, genome, and now endosymbiotic host tissue or cell levels.

Global patterns in genomic diversity underpinning the evolution of insecticide resistance in the aphid crop pest Myzus persicae.

The aphid Myzus persicae is a destructive agricultural pest that displays an exceptional ability to develop resistance to both natural and synthetic insecticides. To investigate the evolution of resistance in this species we generated a chromosome-scale genome assembly and living panel of >110 fully sequenced globally sampled clonal lines. Our analyses reveal a remarkable diversity of resistance mutations segregating in global populations of M. persicae. We show that the emergence and spread of these mechanisms is influenced by host-plant associations, uncovering the widespread co-option of a host-plant adaptation that also offers resistance against synthetic insecticides. We identify both the repeated evolution of independent resistance mutations at the same locus, and multiple instances of the evolution of novel resistance mechanisms against key insecticides. Our findings provide fundamental insights into the genomic responses of global insect populations to strong selective forces, and hold practical relevance for the control of pests and parasites.

Further evidence that mechanisms of host/symbiont integration are dissimilar in the maternal versus embryonic Acyrthosiphon pisum bacteriome.

BACKGROUND: Host/symbiont integration is a signature of evolutionarily ancient, obligate endosymbioses. However, little is known about the cellular and developmental mechanisms of host/symbiont integration at the molecular level. Many insects possess obligate bacterial endosymbionts that provide essential nutrients. To advance understanding of the developmental and metabolic integration of hosts and endosymbionts, we track the localization of a non-essential amino acid transporter, ApNEAAT1, across asexual embryogenesis in the aphid, Acyrthosiphon pisum. Previous work in adult bacteriomes revealed that ApNEAAT1 functions to exchange non-essential amino acids at the A. pisum/Buchnera aphidicola symbiotic interface. Driven by amino acid concentration gradients, ApNEAAT1 moves proline, serine, and alanine from A. pisum to Buchnera and cysteine from Buchnera to A. pisum. Here, we test the hypothesis that ApNEAAT1 is localized to the symbiotic interface during asexual embryogenesis. RESULTS: During A. pisum asexual embryogenesis, ApNEAAT1 does not localize to the symbiotic interface. We observed ApNEAAT1 localization to the maternal follicular epithelium, the germline, and, in late-stage embryos, to anterior neural structures and insect immune cells (hemocytes). We predict that ApNEAAT1 provisions non-essential amino acids to developing oocytes and embryos, as well as to the brain and related neural structures. Additionally, ApNEAAT1 may perform roles related to host immunity. CONCLUSIONS: Our work provides further evidence that the embryonic and adult bacteriomes of asexual A. pisum are not equivalent. Future research is needed to elucidate the developmental time point at which the bacteriome reaches maturity.

The green peach aphid gut contains host plant microRNAs identified by comprehensive annotation of Brassica oleracea small RNA data.

Like all organisms, aphids, plant sap-sucking insects that house a bacterial endosymbiont called Buchnera, are members of a species interaction network. Ecological interactions across such networks can result in phenotypic change in network members mediated by molecular signals, like microRNAs. Here, we interrogated small RNA data from the aphid, Myzus persicae, to determine the source of reads that did not map to the aphid or Buchnera genomes. Our analysis revealed that the pattern was largely explained by reads that mapped to the host plant, Brassica oleracea, and a facultative symbiont, Regiella. To start elucidating the function of plant small RNA in aphid gut, we annotated 213 unique B. oleracea miRNAs; 32/213 were present in aphid gut as mature and star miRNAs. Next, we predicted targets in the B. oleracea and M. persicae genomes for these 32 plant miRNAs. We found that plant targets were enriched for genes associated with transcription, while the distribution of targets in the aphid genome was similar to the functional distribution of all genes in the aphid genome. We discuss the potential of plant miRNAs to regulate aphid gene expression and the mechanisms involved in processing, export and uptake of plant miRNAs by aphids.

Trading amino acids at the aphid-Buchnera symbiotic interface.

Plant sap-feeding insects are widespread, having evolved to occupy diverse environmental niches despite exclusive feeding on an impoverished diet lacking in essential amino acids and vitamins. Success depends exquisitely on their symbiotic relationships with microbial symbionts housed within specialized eukaryotic bacteriocyte cells. Each bacteriocyte is packed with symbionts that are individually surrounded by a host-derived symbiosomal membrane representing the absolute host-symbiont interface. The symbiosomal membrane must be a dynamic and selectively permeable structure to enable bidirectional and differential movement of essential nutrients, metabolites, and biosynthetic intermediates, vital for growth and survival of host and symbiont. However, despite this crucial role, the molecular basis of membrane transport across the symbiosomal membrane remains unresolved in all bacteriocyte-containing insects. A transport protein was immunolocalized to the symbiosomal membrane separating the pea aphid Acyrthosiphon pisum from its intracellular symbiont Buchnera aphidicola The transporter, A. pisum nonessential amino acid transporter 1, or ApNEAAT1 (gene: ACYPI008971), was characterized functionally following heterologous expression in Xenopus oocytes, and mediates both inward and outward transport of small dipolar amino acids (serine, proline, cysteine, alanine, glycine). Electroneutral ApNEAAT1 transport is driven by amino acid concentration gradients and is not coupled to transmembrane ion gradients. Previous metabolite profiling of hemolymph and bacteriocyte, alongside metabolic pathway analysis in host and symbiont, enable prediction of a physiological role for ApNEAAT1 in bidirectional host-symbiont amino acid transfer, supplying both host and symbiont with indispensable nutrients and biosynthetic precursors to facilitate metabolic complementarity.

mTOR Complex 1 Implicated in Aphid/Buchnera Host/Symbiont Integration.

Obligate nutritional endosymbioses are arguably the most intimate of all interspecific associations. While many insect nutritional endosymbioses are well studied, a full picture of how two disparate organisms, a bacterial endosymbiont and a eukaryotic host, are integrated is still lacking. The mTOR pathway is known to integrate nutritional conditions with cell growth and survival in eukaryotes. Characterization and localization of amino acid transporters in aphids suggest the mTOR pathway as a point of integration between an aphid host and its amino acid-provisioning endosymbiont Buchnera aphidicola The mTOR pathway is unannotated in aphids and unstudied in any nutritional endosymbiosis. We annotated mTOR pathway genes in two aphid species, Acyrthosiphon pisum and Myzus persicae, using both BLASTp searches and Hidden Markov Models. Using previously collected RNAseq data we constructed new reference transcriptomes for bacteriocyte, gut, and whole insect tissue for three lines of M. persicae Annotation of the mTOR pathway identified homologs of all known invertebrate mTOR genes in both aphid species with some duplications. Differential expression analysis showed that genes specific to the amino acid-sensitive mTOR Complex 1 were more highly expressed in bacteriocytes than genes specific to the amino acid-insensitive mTOR Complex 2. Almost all mTOR genes involved in sensing amino acids showed higher expression in bacteriocytes than in whole insect tissue. When compared to gut, the putative glutamine/arginine sensing transporter ACYPI000333, an ortholog of SLC38A9, showed 6.5 times higher expression in bacteriocytes. Our results suggest that the mTOR pathway may be functionally important in mediating integration of Buchnera into aphid growth and reproduction.

microRNA regulation in an ancient obligate endosymbiosis.

Although many insects are associated with obligate bacterial endosymbionts, the mechanisms by which these host/endosymbiont associations are regulated remain mysterious. While microRNAs (miRNAs) have been recently identified as regulators of host/microbe interactions, including host/pathogen and host/facultative endosymbiont interactions, the role miRNAs may play in mediating host/obligate endosymbiont interactions is virtually unknown. Here, we identified conserved miRNAs that potentially mediate symbiotic interactions between aphids and their obligate endosymbiont, Buchnera aphidicola. Using small RNA sequence data from Myzus persicae and Acyrthosiphon pisum, we annotated 93 M. persicae and 89 A. pisum miRNAs, among which 69 were shared. We found 14 miRNAs that were either highly expressed in aphid bacteriome, the Buchnera-housing tissue, or differentially expressed in bacteriome vs. gut, a non-Buchnera-housing tissue. Strikingly, 10 of these 14 miRNAs have been implicated previously in other host/microbe interaction studies. Investigating the interaction networks of these miRNAs using a custom computational pipeline, we identified 103 miRNA::mRNA interactions shared between M. persicae and A. pisum. Functional annotation of the shared mRNA targets revealed only two over-represented cluster of orthologous group categories: amino acid transport and metabolism, and signal transduction mechanisms. Our work supports a role for miRNAs in mediating host/symbiont interactions between aphids and their obligate endosymbiont Buchnera. In addition, our results highlight the probable importance of signal transduction mechanisms to host/endosymbiont coevolution.

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species.

BACKGROUND: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. RESULTS: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. CONCLUSIONS: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

Amino acid transporters implicated in endocytosis of Buchnera during symbiont transmission in the pea aphid.

BACKGROUND: Many insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration. RESULTS: We present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos. CONCLUSIONS: We venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development.

Gene Family Expansions in Aphids Maintained by Endosymbiotic and Nonsymbiotic Traits.

Facilitating the evolution of new gene functions, gene duplication is a major mechanism driving evolutionary innovation. Gene family expansions relevant to host/symbiont interactions are increasingly being discovered in eukaryotes that host endosymbiotic microbes. Such discoveries entice speculation that gene duplication facilitates the evolution of novel, endosymbiotic relationships. Here, using a comparative transcriptomic approach combined with differential gene expression analysis, we investigate the importance of endosymbiosis in retention of amino acid transporter paralogs in aphid genomes. To pinpoint the timing of amino acid transporter duplications we inferred gene phylogenies for five aphid species and three outgroups. We found that while some duplications arose in the aphid common ancestor concurrent with endosymbiont acquisition, others predate aphid divergence from related insects without intracellular symbionts, and still others appeared during aphid diversification. Interestingly, several aphid-specific paralogs have conserved enriched expression in bacteriocytes, the insect cells that host primary symbionts. Conserved bacteriocyte enrichment suggests that the transporters were recruited to the aphid/endosymbiont interface in the aphid common ancestor, consistent with a role for gene duplication in facilitating the evolution of endosymbiosis in aphids. In contrast, the temporal variability of amino acid transporter duplication indicates that endosymbiosis is not the only trait driving selection for retention of amino acid transporter paralogs in sap-feeding insects. This study cautions against simplistic interpretations of the role of gene family expansion in the evolution of novel host/symbiont interactions by further highlighting that multiple complex factors maintain gene family paralogs in the genomes of eukaryotes that host endosymbiotic microbes.

Ontogenetic differences in localization of glutamine transporter ApGLNT1 in the pea aphid demonstrate that mechanisms of host/symbiont integration are not similar in the maternal versus embryonic bacteriome.

BACKGROUND: Obligate intracellular symbionts of insects are metabolically and developmentally integrated with their hosts. Typically, reproduction fails in many insect nutritional endosymbioses when host insects are cured of their bacterial symbionts, and yet remarkably little is known about the processes that developmentally integrate host and symbiont. Here in the best studied insect obligate intracellular symbiosis, that of the pea aphid, Acyrthosiphon pisum, with the gammaproteobacterium Buchnera aphidicola, we tracked the expression and localization of amino acid transporter ApGLNT1 gene products during asexual embryogenesis. Recently being characterized as a glutamine transporter, ApGLNT1 has been proposed to be a key regulator of amino acid biosynthesis in A. pisum bacteriocytes. To determine when this important mediator of the symbiosis becomes expressed in aphid embryonic bacteriocytes, we applied whole-mount in situ hybridization and fluorescent immunostaining with a specific anti-ApGLNT1 antibody to detect the temporal and spatial expression of ApGLNT1 gene products during asexual embryogenesis. RESULTS: During embryogenesis, ApGLNT1 mRNA and protein localize to the follicular epithelium that surrounds parthenogenetic viviparous embryos, where we speculate that it functions to supply developing embryos with glutamine from maternal hemolymph. Unexpectedly, in the embryonic bacteriome ApGLNT1 protein does not localize to the membrane of bacteriocytes, a pattern that leads us to conclude that the regulation of amino acid metabolism in the embryonic bacteriome mechanistically differs from that in the maternal bacteriome. Paralleling our earlier report of punctate cytoplasmic localization of ApGLNT1 in maternal bacteriocytes, we find ApGLNT1 protein localizing as cytoplasmic puncta throughout development in association with Buchnera. CONCLUSIONS: Our work that documents ontogenetic shifts in the localization of ApGLNT1 protein in the host bacteriome demonstrates that maternal and embryonic bacteriomes are not equivalent. Significantly, the persistent punctate cytoplasmic localization of ApGLNT1 in association with Buchnera in embryos prior to bacteriocyte formation and later in both embryonic and maternal bacteriomes suggests that ApGLNT1 plays multiple roles in this symbiosis, roles that include amino acid transport and possibly nutrient sensing.

Proton-dependent glutamine uptake by aphid bacteriocyte amino acid transporter ApGLNT1.

Aphids house large populations of the gammaproteobacterial symbiont Buchnera aphidicola in specialized bacteriocyte cells. The combined biosynthetic capability of the holobiont (Acyrthosiphon pisum and Buchnera) is sufficient for biosynthesis of all twenty protein coding amino acids, including amino acids that animals alone cannot synthesize; and that are present at low concentrations in A. pisum's plant phloem sap diet. Collaborative holobiont amino acid biosynthesis depends on glutamine import into bacteriocytes, which serves as a nitrogen-rich amino donor for biosynthesis of other amino acids. Recently, we characterized A. pisum glutamine transporter 1 (ApGLNT1), a member of the amino acid/auxin permease family, as the dominant bacteriocyte plasma membrane glutamine transporter. Here we show ApGLNT1 to be structurally and functionally related to mammalian proton-dependent amino acid transporters (PATs 1-4). Using functional expression in Xenopus laevis oocytes, combined with two-electrode voltage clamp electrophysiology we demonstrate that ApGLNT1 is electrogenic and that glutamine induces large inward currents. ApGLNT1 glutamine induced currents are dependent on external glutamine concentration, proton (H+) gradient across the membrane, and membrane potential. Based on these transport properties, ApGLNT1-mediated glutamine uptake into A. pisum bacteriocytes can be regulated by changes in either proton gradients across the plasma membrane or membrane potential.

Signatures of host/symbiont genome coevolution in insect nutritional endosymbioses.

The role of symbiosis in bacterial symbiont genome evolution is well understood, yet the ways that symbiosis shapes host genomes or more particularly, host/symbiont genome coevolution in the holobiont is only now being revealed. Here, we identify three coevolutionary signatures that characterize holobiont genomes. The first signature, host/symbiont collaboration, arises when completion of essential pathways requires host/endosymbiont genome complementarity. Metabolic collaboration has evolved numerous times in the pathways of amino acid and vitamin biosynthesis. Here, we highlight collaboration in branched-chain amino acid and pantothenate (vitamin B5) biosynthesis. The second coevolutionary signature is acquisition, referring to the observation that holobiont genomes acquire novel genetic material through various means, including gene duplication, lateral gene transfer from bacteria that are not their current obligate symbionts, and full or partial endosymbiont replacement. The third signature, constraint, introduces the idea that holobiont genome evolution is constrained by the processes governing symbiont genome evolution. In addition, we propose that collaboration is constrained by the expression profile of the cell lineage from which endosymbiont-containing host cells, called bacteriocytes, are derived. In particular, we propose that such differences in bacteriocyte cell lineage may explain differences in patterns of host/endosymbiont metabolic collaboration between the sap-feeding suborders Sternorrhyncha and Auchenorrhynca. Finally, we review recent studies at the frontier of symbiosis research that are applying functional genomic approaches to characterization of the developmental and cellular mechanisms of host/endosymbiont integration, work that heralds a new era in symbiosis research.

Amino acid transporter expansions associated with the evolution of obligate endosymbiosis in sap-feeding insects (Hemiptera: sternorrhyncha).

BACKGROUND: Mutualistic obligate endosymbioses shape the evolution of endosymbiont genomes, but their impact on host genomes remains unclear. Insects of the sub-order Sternorrhyncha (Hemiptera) depend on bacterial endosymbionts for essential amino acids present at low abundances in their phloem-based diet. This obligate dependency has been proposed to explain why multiple amino acid transporter genes are maintained in the genomes of the insect hosts. We implemented phylogenetic comparative methods to test whether amino acid transporters have proliferated in sternorrhynchan genomes at rates grater than expected by chance. RESULTS: By applying a series of methods to reconcile gene and species trees, inferring the size of gene families in ancestral lineages, and simulating the null process of birth and death in multi-gene families, we uncovered a 10-fold increase in duplication rate in the AAAP family of amino acid transporters within Sternorrhyncha. This gene family expansion was unmatched in other closely related clades lacking endosymbionts that provide essential amino acids. CONCLUSIONS: Our findings support the influence of obligate endosymbioses on host genome evolution by both inferring significant expansions of gene families involved in symbiotic interactions, and discovering increases in the rate of duplication associated with multiple emergences of obligate symbiosis in Sternorrhyncha.

Biased transmission of sex chromosomes in the aphid Myzus persicae is not associated with reproductive mode.

Commonly, a single aphid species exhibits a wide range of reproductive strategies including cyclical parthenogenesis and obligate parthenogenesis. Sex determination in aphids is chromosomal; females have two X chromosomes, while males have one. X chromosome elimination at male production is generally random, resulting in equal representation of both X chromosomes in sons. However, two studies have demonstrated deviations from randomness in some lineages. One hypothesis to account for such deviations is that recessive deleterious mutations accumulate during bouts of asexual reproduction and affect male viability, resulting in overrepresentation of males with the least deleterious of the two maternal X chromosomes. This hypothesis results in a testable prediction: X chromosome transmission bias will increase with time spent in the asexual phase and should therefore be most extreme in the least sexual aphid life cycle class. Here we test this prediction in Myzus persicae. We used multiple heterozygous X-linked microsatellite markers to screen 1085 males from 95 lines of known life cycle. We found significant deviations from equal representation of X chromosomes in 15 lines; however, these lines included representatives of all life cycles. Our results are inconsistent with the hypothesis that deviations from randomness are attributable to mutation accumulation.

A substrate ambiguous enzyme facilitates genome reduction in an intracellular symbiont.

BACKGROUND: Genome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism. RESULTS: We investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme. CONCLUSIONS: Earlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.

Dynamic recruitment of amino acid transporters to the insect/symbiont interface.

Symbiosis is well known to influence bacterial symbiont genome evolution and has recently been shown to shape eukaryotic host genomes. Intriguing patterns of host genome evolution, including remarkable numbers of gene duplications, have been observed in the pea aphid, a sap-feeding insect that relies on a bacterial endosymbiont for amino acid provisioning. Previously, we proposed that gene duplication has been important for the evolution of symbiosis based on aphid-specific gene duplication in amino acid transporters (AATs), with some paralogs highly expressed in the cells housing symbionts (bacteriocytes). Here, we use a comparative approach to test the role of gene duplication in enabling recruitment of AATs to bacteriocytes. Using genomic and transcriptomic data, we annotate AATs from sap-feeding and non sap-feeding insects and find that, like aphids, AAT gene families have undergone independent large-scale gene duplications in three of four additional sap-feeding insects. RNA-seq differential expression data indicate that, like aphids, the sap-feeding citrus mealybug possesses several lineage-specific bacteriocyte-enriched paralogs. Further, differential expression data combined with quantitative PCR support independent evolution of bacteriocyte enrichment in sap-feeding insect AATs. Although these data indicate that gene duplication is not necessary to initiate host/symbiont amino acid exchange, they support a role for gene duplication in enabling AATs to mediate novel host/symbiont interactions broadly in the sap-feeding suborder Sternorrhyncha. In combination with recent studies on other symbiotic systems, gene duplication is emerging as a general pattern in host genome evolution.