Signatures of host/symbiont genome coevolution in insect nutritional endosymbioses

Abstract

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.

Many insects have established persistent, intimate associations with vertically transmitted microbial symbionts. These ancient, commonly intracellular symbionts are, owing to their greatly reduced, gene-poor genomes, uncultivable outside their hosts. Although the whole-genome sequences of obligate insect endosymbionts continue to yield novel insight into genome evolution following adoption of an obligate intracellular lifestyle (e.g., ref. 1), it is the recent partnering of these symbiont genome sequences with those of their insect hosts that fundamentally shifts conceptualization of “the organism,” propelling us into a new era in symbiosis research.

The first paired insect host/endosymbiont genome available was that of the pea aphid, Acyrthosiphon pisum and its bacterial endosymbiont, Buchnera aphidicola (2, 3). The second was that of the human body louse, Pediculus humanus humanus and its primary bacterial endosymbiont Candidatus Riesia pediculicola (4), quickly followed by the carpenter ant, Camponotus floridanus (5) and its endosymbiont Candidatus Blochmannia floridanus (6). Other insect holobiont genomes now include the citrus mealybug, Planococcus citri with its dual endosymbionts (7, 8); the hackberry petiole gall psyllid Pachypsylla venusta with its endosymbiont Candidatus Carsonella ruddii (9); and the tsetse fly, Glossina morsitans (10) with its endosymbiont Wigglesworthia glossinidia (11, 12). This recent accumulation of holobiont genomes facilitates elucidation of the patterns that characterize coevolution in these ancient, intimate symbiotic associations. Our purpose here is to highlight three signatures of genome coevolution across available holobiont genomes and to draw attention to work at the frontier of symbiosis research that elucidates mechanisms of holobiont regulation and integration.

Three Signatures: Collaboration, Acquisition, and Constraint

Holobiont genome evolution is characterized by patterns of collaboration, acquisition, and constraint. Coevolution typically features host/endosymbiont collaboration on completion of critical metabolic pathways—a set of pathways that is similar across taxa, apparently constrained by eukaryotic host gene repertoire and yet, concurrently, holobiont genome evolution is dynamic. Dynamic features include acquisition of novel genomic material like duplicate genes, genes acquired by lateral gene transfer that enhance collaboration, and acquisition of coprimary symbionts or even new primary symbionts by symbiont replacement.

Collaboration

Long before it was possible to elucidate the metabolic repertoire of an organism by sequencing its DNA, researchers established the metabolic basis of many insect nutritional symbioses experimentally by quantifying the growth and fecundity of insects manipulated in a diversity of ways that included some combination of endosymbiont removal and diet manipulations (13). Such analyses established that endosymbionts provision host insects with dietary components lacking or at low availability in their diets. Thus, at the beginning of the genomic revolution it was understood that hosts supply endosymbionts with metabolic precursors that endosymbionts metabolically transform into host-required dietary components. Notably, from an organismal perspective, insect nutritional symbioses in pregenome times were partnerships between two discrete organisms: the host and the endosymbiont. Endosymbiont whole-genome sequencing affirmed and continues to affirm the nutritional role played by symbionts, whereas host genome and transcriptome sequencing reveals a complex portrait of the nature and extent of host/endosymbiont metabolic complementarity.

Currently the most well-documented examples of host/endosymbiont metabolic collaboration involve the biosynthesis of essential amino acids in plant sap-feeding insects (2, 7–9, 14–16). In most cases, holobiont metabolism has been reconstructed using the whole-genome sequence of the symbiont coupled with a host bacteriome transcriptome (the bacteriome is the host organ composed of host bacteriocyte cells that house the endosymbionts) (Fig. 1) and a partial host genome. In the case of the pea aphid, metabolic reconstruction leveraged the complete genome of both symbiont and host (2, 14). The many similarities and differences in the collaborative amino acid biosynthesis of plant sap-feeding holobionts have recently been reviewed by Sloan et al. (9) and Hansen and Moran (17). Therefore, here we illustrate the collaborative signature of host/symbiont genome coevolution by drawing attention to one set of amino acid biosynthesis pathways, those for biosynthesis of the branched-chain amino acids. Biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine, involves host/endosymbiont metabolic collaboration in both A. pisum (14, 15, 16) and P. citri (7, 8). The endosymbionts of both insects have lost the transaminase encoded by ilvE, an enzyme (E.C. 2.6.1.42) that mediates the terminal reaction in the biosynthesis of all three branched-chain amino acids. However, the genomes of both host insects encode branched-chain amino acid transaminase (BCA). Thus, in both symbioses, completion of branched-chain amino acid biosynthesis has been hypothesized to occur through host/endosymbiont metabolic collaboration (7, 14). Recently, Russell et al. (16), partially validated the model of BCA complementation of leucine biosynthesis in A. pisum by demonstrating that release of leucine from isolated Buchnera cells increased in the presence of host cellular fractions containing BCA. Biosynthesis of the branched-chain amino acids serves as just one example of host/endosymbiont metabolic collaboration. Other examples include phenylalanine (9, 14, 17), methionine (8, 9, 14), and arginine (8, 9) biosynthesis.

Fig. 1.

Organization of endosymbionts in insect hosts. Typically enveloped by a host membrane (green) within the cytoplasm of specialized host bacteriocyte cells, insect nutritional endosymbionts (purple) are maternally transmitted and integrated into host development by currently unknown mechanisms. Individual bacteriocyte cells house thousands to tens of thousands of symbionts. Many bacteriocyte cells aggregate to form an organ called the bacteriome. Bacteriomes are typically paired and relatively large.

Another example of host/endosymbiont metabolic collaboration comes from examination of vitamin biosynthesis pathways. Recent work in A. pisum extends evidence of host/endosymbiont metabolic collaboration to include pantothenate (vitamin B5) biosynthesis (18). Pantothenate biosynthesis requires a supply of β-alanine. Buchnera APS has lost panD (encoding E.C. 4.1.1.11) and with it, the ability to synthesize β-alanine. In contrast, A. pisum, like most insects, retains two pathways for synthesis of β-alanine. Of these two pathways, the expression of the uracil degradation pathway is up-regulated in bacteriocytes (the cells that house the endosymbionts) and the expression of the alternate pathway is down-regulated. Price and Wilson (18) propose that β-alanine biosynthesis by A. pisum in bacteriocytes serves to provision Buchnera and facilitate maintenance of a functional pantothenate biosynthesis pathway. Given the common patterns of collaboration in amino acid biosynthesis across holobionts (reviewed in refs. 9 and 17), it is notable that the endosymbionts of P. citri (8), Homalodisca vitripennis (19), P. humanus (4), and G. morsitans (12), like Buchnera APS, have lost panD and therefore the ability to synthesize β-alanine. At the same time their role in pantothenate biosynthesis is strongly supported by retention of panC, a gene encoding pantothenate synthetase (E.C. 6.3.2.1) that functions only in pantothenate biosynthesis (Fig. 2) (18). Thus, we venture that examination of the host genomes and transcriptomes of these four endosymbionts will demonstrate that pantothenate biosynthesis is indeed functional in these holobionts as a result of host/endosymbiont metabolic collaboration.

Fig. 2.

Patterns of gene retention and loss in the pantothenate biosynthesis pathway from insect obligate nutritional symbionts. Symbionts from all five hosts have lost the ability to synthesize β-alanine, whereas all retain panC, encoding pantothenate synthetase (E.C. 6.3.2.1), a gene that functions only in pantothenate biosynthesis.

It is clear that metabolic collaboration is a fundamental signature of host/symbiont genome coevolution. However, the extent to which patterns of metabolic collaboration in holobionts result from convergence and not shared ancestry remains to be determined. Distinguishing between traits shared by convergence and those shared by common ancestry requires character state information for a broad range of closely related taxa and unfortunately such data are not currently available. That said, on the basis of the data that are available, we venture that metabolic collaboration has evolved independently in multiple metabolic pathways in multiple insect lineages. For example, the human body louse, P. humanus and the pea aphid, A. pisum belong to different insect orders and yet both require host/endosymbiont collaboration for maintenance of functional pantothenate biosynthesis pathways. As described above, A. pisum (18) and likely P. humanus depend on host supply of β-alanine for symbiont-mediated biosynthesis of pantothenate (Fig. 2). Further, pantothenate biosynthesis probably requires additional host complementation in P. humanus for synthesis of the metabolic intermediate 2-oxoisovalerate, a metabolite synthesized in A. pisum by the endosymbiont Buchnera. Intriguingly, the difference in 2-oxoisovalerate biosynthesis in A. pisum and P. humanus likely results from selection to maintain amino acid biosynthesis pathways in Buchnera, the symbiont of plant sap-feeder A. pisum, and loss of these same pathways in Riesia, the symbiont of blood-feeder P. humanus (4). This example aside, definitive determination of what host/symbiont collaboration patterns result from convergence vs. descent unavoidably requires generation and analysis of an extensive dataset that does not yet exist, a dataset that may be some time in coming.

Common patterns of collaborative maintenance of functional metabolism continue to be discovered across systems (e.g., refs. 7–9, 14, 17) and yet, common patterns are not always found, even when genomic potential for the evolution of collaboration exists. For example, all sap-feeding insects depend on endosymbiont supply of the branched-chain amino acids that are collaboratively synthesized in A. pisum (14, 15, 16) and P. citri (7, 8) but not collaboratively synthesized in sap-feeding auchenorrhyncans (20, 21) or the psyllid P. venusta (9). We propose below in the context of the third signature that such differences in patterns of collaboration across taxa are governed by cellular and developmental constraints.

Acquisition

The second signature of host/symbiont genome coevolution is acquisition; holobiont coevolution in nutritional insect symbioses spans hundreds of millions of years (22–25) and yet recent work in many holobionts demonstrates that their genomes are dynamic. Three mechanisms have been documented that contribute to a genome-level change in insect nutritional symbioses. These include gene duplication, lateral gene transfer, and partial or full symbiont replacement.

Gene duplication events facilitate refinement of existing function, the evolution of new spatial and temporal gene expression patterns, and even the evolution of new gene functions (26–28). Recent work in insect nutritional endosymbionts has focused on the evolution of nutrient amino acid transporters in the genomes of insects that feed on plant sap (29–32). Those studies find that the evolutionary history of amino acid transporter genes in plant sap-feeding insects is dynamic with respect to both duplication events and the recruitment of duplicated genes to the host/symbiont interface (31). The dynamic evolution of amino acid transporters in these insects, including some very recent duplications in aphids (32), demonstrates that despite millions of years of host/endosymbiont coevolution, host genomes are in flux (Fig. 3). Functional genomics work across sap-feeding insects is necessary to determine the extent to which these changes in amino acid transporter evolution contribute to host/endosymbiont integration.

Fig. 3.

Insects with sequenced host and symbiont genomes. Shown is the phylogeny of representative insect hosts based on Misof et al. (66), Cryan and Urban (67), and Dahan et al. (32). Insect hosts and their respective symbionts appear at the tips of the branches, with symbiont classification represented by text color. Superscripts “G” (genome) and “T” (transcriptome) denote the genomic resources available for each insect and symbiont. Four genomic characters represented by different symbols (Inset) are mapped onto branches indicating the most parsimonious explanation for their distribution among holosymbionts. “Host metabolic complementation” refers to presence of endogenous host genes complementing missing symbiont genes, with insect gene names listed below the character symbol. “Host amino acid transporter expansion” refers to the evolution of lineage-specific duplications in amino acid transporters, with Drosophila melanogaster orthologs of expanded transporter listed beside the symbol. The D. melanogaster gene slimfast is listed on all three sap-feeding insect lineages because its ortholog expanded independently in each of these insects (9, 31). “LGT to host genome (metabolism gene)” refers to host genome acquisition of bacterial genes that complement missing symbiont genes with roles in metabolism; the names of host-encoded bacterial genes are listed beside the symbol. “LGT to host genome (nonmetabolism gene)” refers to host genome acquisition of bacterial genes that complement missing symbiont genes with nonmetabolic roles.

Lateral transfer of bacterial genes to host genomes has the potential to relieve coevolutionary constraints in a marvelous way. Such lateral gene transfer in the context of a nutritional endosymbiosis was first reported in A. pisum (33, 34). Whole-genome analysis of A. pisum identified the transfer of 12 genes or gene fragments from bacteria to the insect genome (34). Of those 12, 7 were highly expressed in bacteriocyte cells but none were implicated in amino acid or vitamin biosynthesis. Recent work by Husnik et al. (8) in the mealybug P. citri and Sloan et al. (9) in the hackberry petiole gall psyllid P. venusta found that metabolism central to symbiotic function depends on host-encoded genes of bacterial origin that were acquired laterally from multiple bacterial lineages, together revealing an important additional mechanism facilitating holobiont integration (Fig. 3). Given the newness of the discovery of lateral gene transfer events that facilitate host/endosymbiont metabolic collaboration, the impact and extent of lateral gene transfer on host/endosymbiont integration remain to be fully appreciated.

Partial or full endosymbiont replacement by previously facultative insect symbionts is a third mode by which holobionts gain new genetic material. Despite the fact that symbiont genome evolution is characterized by genome degradation, many endosymbionts have coevolved with their insect hosts since ancient times (35). When bacteria become obligate symbionts, important population genetic parameters immediately change. For example, bacterial symbionts, unlike their free-living relatives, experience relaxed selection and greatly reduced population size, resulting in elevated genetic drift (35–37). As a consequence of elevated genetic drift and small population size, mildly deleterious mutations become fixed at a higher rate than they would in a large population. Further, because of an absence of sex and recombination, any symbiont population can carry a mutation load only equal to or higher than that of its least-loaded lines; this feature of evolution in small, nonrecombining populations is know as Muller’s ratchet (38). Over evolutionary time, Muller’s ratchet results in endosymbionts having highly reduced and degraded genomes. Despite the degenerative nature of endosymbiont genome evolution, there is strong selection for symbionts to retain genes essential to performing their metabolic duties, except when another symbiont with the same metabolic capacity infects the host. Mechanistically, then, the acquisition of facultative symbionts both relaxes selective pressures on the maintenance of essential genes in the primary symbiont and provides opportunity for the replacement or complementation of ancestral endosymbiont function or functions that are lost as a result of relaxed selection. In such cases, facultative symbionts become obligate, thereby facilitating maintenance of functions that have become eroded over millions of years by Muller’s ratchet (38).

Full symbiont replacement and partial symbiont replacement—i.e., the occurrence of coprimary symbionts—are common features of holobiont evolution. Examples of full symbiont replacement in sap-feeding nutritional symbioses include aphids with a secondarily acquired fungal symbiont (39) and the mealybug genus Rastrococcus, in which the ancestral, betaproteobacterial symbiont Tremblaya was replaced by a Bacteroidetes (40). Further, in the Auchenorrhyncha, which typically have two primary symbionts, one symbiont (Sulcia) has been maintained through evolution whereas its coprimary symbiont has been replaced many times such that different auchenorrhynchan lineages have alphaproteobacterial, betaproteobacterial, gammaproteobacterial, or yeast-like symbionts (20, 21, 41) (Fig. 3). Likewise, there are many examples of coprimary symbionts in aphids, mealybugs, and auchenorrhynchans that partition metabolic roles (19–21) and sometimes even provide genes that complement missing genes from the genomes of their cosymbiont (7, 42, 43). Symbiont replacement can even arise when an ancestral symbiont diverges into two interdependent lineages (1). The replacement or complementation of a primary endosymbiont by a more recently acquired (or derived) symbiont has the potential to functionally “reset” genes and pathways that have been eroded by mutation accumulation over evolutionary time.

Constraint

Emerging as the third signature of coevolution in insect nutritional endosymbioses, constraint influences the evolution of metabolic collaboration, the acquisition of bacterial genes by host genomes, and symbiont replacement. The role of constraint in metabolic collaboration becomes clear in the observation that by convergence and not by descent, the same insect genes in different holobionts are engaged in host/symbiont metabolic collaboration, complementing the same symbiont gene losses. Opportunities to evolve collaborative biosynthesis are constrained by the gene content of host genomes. Additionally, we propose that the coevolutionary potential of an endosymbiosis is constrained by the cell type that gave rise to the bacteriome in each taxon. Although all nucleated cell types contain the complete host genome, all cell types do not express all genes. Therefore, differences in basal expression of the cell lineage that gives rise to the bacteriome in each taxon will constrain patterns of holobiont coevolution. In fact, as we discuss below, we propose that cell lineage differences may explain differences in collaborative branched-chain amino acid biosynthesis among the Sternorrhyncha and Auchenorrhynca (Fig. 3).

By convergence, not by descent, metabolic pathways are integrated so that completion of a collaborative pathway requires contribution of gene products from both symbiont and host. We described examples of collaborative biosynthesis of amino acids and vitamins in the first section; here we argue that patterns of collaborative biosynthesis have evolved independently in multiple insect lineages because they are constrained by host gene repertoire. When endosymbiont genomes encode genes that are functionally redundant with host genes (or coresident symbiont genes), endosymbionts experience relaxed selection to maintain their functionally redundant genes. Given the process of gene loss in symbiont genomes, endosymbionts are more likely to lose a gene if it is complemented by the host genome. Thus, it is not surprising that within Sternorrhyncha, the biosynthesis of the branched-chain amino acids, as well as phenylalanine and methionine, requires host complementation at the same steps, steps mediated by enzymes broadly conserved across the Metazoa. Although shared ancestry precludes our ability to confidently distinguish between convergence and homology in shared sternorrhynchan metabolic complementation, other lines of evidence support host gene repertoire as a constraining factor in symbiont gene loss. For example, BCA independently enabled the evolution of metabolic collaboration in different pathways and in different symbiotic contexts. In Sternorrhyncha, BCA mediates the terminal step in branched-chain amino acid biosynthesis, complementing the missing symbiont gene, ilvE (14, 16). On the basis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, we propose that in the divergent blood feeders P. humanus and G. morsitans, BCA also complements the missing symbiont ilvE, this time mediating conversion of valine to pantoate in panthothenate biosynthesis (4, 12, 44). These examples demonstrate that at least three times, symbionts have independently lost the same gene (ilvE) whose function was complemented by a conserved host gene, indicating that symbiont gene loss is indeed constrained by host gene repertoire. Additional evidence for a role of host gene content in shaping symbiont gene content is illustrated by symbiont gene losses when hosts have acquired functionally equivalent genes from bacteria through lateral gene transfer (8, 9). Thus, as lateral gene transfer to the host genome expands host gene repertoire, it additionally relaxes selective constraints on symbiont gene content, providing further opportunities for host/endosymbiont metabolic collaboration.

Apart from host gene repertoire, another factor likely constraining holobiont genome evolution is the gene expression profile of the cells that give rise to bacteriomes. This constraint is evident in symbiont retention of essential metabolic genes that are functionally redundant with host-encoded genes. For example, returning again to branched-chain amino acid biosynthesis, P. citri and A. pisum both evolved metabolic collaboration in branched-chain amino acid biosynthesis, where the terminal step is carried out by the insect gene BCA, but branched-chain amino acid biosynthesis is intact in symbionts of P. venusta, other psyllids, and all auchenorrhynchan sap feeders studied to date. This difference in collaboration is not driven by host gene content, as BCA is encoded in the genome of P. venusta (9) and transcribed in the cicada Diceroprocta semicincta (31). The presence of BCA in insects whose symbionts have intact branched-chain amino acid biosynthesis demonstrates that host gene content by itself is not sufficient to enable the evolution of metabolic collaboration. The evolution of metabolic collaboration requires expression of both complementary host metabolism genes and host and/or symbiont transporters to facilitate movement of metabolites and/or enzymes between host and symbiont. We raise the possibility that the differences we describe above in branched-chain amino acid biosynthesis complementation arise from differences in host gene expression profile in the cells giving rise to the bacteriomes in sternorrhynchans, psyllids, and auchenorrhynchans. We argue this because host/symbiont complementation appears to be a ubiquitous signature of holobiont genome evolution. Thus, if we do not find complementation when host gene content makes complementation possible, the most parsimonious explanation becomes that we do not find complementation because genes necessary for the evolution of complementation are not expressed in symbiont-hosting tissues. That BCA is enriched in P. citri (8) and A. pisum (15) bacteriomes, but is not enriched in the bacteriomes of P. venusta (9), is consistent with the notion that gene expression in symbiont-hosting tissues is an important factor constraining holobiont genome evolution.

In the same way that the accumulation of whole symbiont genomes has solidified understanding of the signatures of bacterial genome evolution following adoption of a symbiotic lifestyle, the accumulation of holobiont genomes will continue to refine understanding of the signatures of host/symbiont genome coevolution. Metabolic collaboration, the acquisition of novel genomic material, and host genomic constraints are emergent features of host/symbiont genome coevolution. Although few in number, the holobiont genomes currently available are facilitating advances at the frontier of symbiosis research, a research frontier focused on elucidating the cellular and developmental mechanisms of host/endosymbiont integration.

Elucidating the Cellular and Developmental Mechanism of Host/Endosymbiont Integration

Compelling questions at the frontier of insect obligate nutritional endosymbiosis research concern mechanisms of holobiont metabolic and developmental integration (45). With respect to metabolic integration, current research focuses on questions of regulation and control of nutrient biosynthesis. Whereas with respect to developmental integration, current research focuses on the cellular localization of novel bacteriocyte-expressed proteins. Notably, many studies at the frontier are characterized by application of tools that facilitate elucidation of the mechanisms of holobiont integration.

Addressing questions about holobiont metabolic and developmental integration requires appreciation of two features typical of most insect obligate nutritional endosymbioses. First, endosymbionts are vertically transmitted and have been for long evolutionary time periods (35). Strict vertical transmission means that the passage of symbionts is predictable and integral to insect development. However, little is known currently about the cell and developmental pathways responsible for nutritional endosymbiont transmission, and the developmental origin of bacteriocyte cells in all taxa is unknown. The second feature typical of most obligate nutritional endosymbioses is the localization of symbionts inside host membranes, inside host bacteriocyte cells, a pattern of containment that creates many metabolically active compartments (Fig. 1). With respect to membranes, recall that membranes are barriers to the free movement of molecules. Thus, membrane barriers have the potential to regulate metabolic inputs from host to symbiont and metabolic outputs from endosymbiont to host.

Two recent studies have addressed the question of metabolic control in the A. pisum/Buchnera symbiosis. Where Buchnera’s weak transcriptional responses to changing nutritional demand (e.g., refs. 46–49) has resulted in the suggestion that control of holobiont metabolism lies with the host (46, 50), recent work by Hansen and Degnan (51) renews the possibility that Buchnera plays an important role in regulating holobiont function. Using label-free mass-spectrometry quantification, Hansen and Degnan (51) found differential protein expression from Buchnera in embryonic vs. maternal bacteriomes. These differences did not result from differences in Buchnera gene expression. Although they found no evidence for differential Buchnera gene expression, they did find over 100 small RNAs and hundreds of untranslated regions that were significantly expressed by Buchnera, some of which matched differentially expressed proteins. Based on their findings, Hansen and Degnan (51) proposed that small RNAs mediate regulation of gene expression at the posttranscriptional level in Buchnera. Taken together, these results reopen the door for recognizing Buchnera as more than a symbiotic puppet under host control.

The second recent study that addressed the question of metabolic control in the A. pisum/Buchnera symbiosis pursued the suggestion by Moran et al. (46) that host control of nonessential amino acid transport to Buchnera could be a mechanism by which aphids regulate endosymbiont metabolism. By functionally characterizing A. pisum amino acid transporters via heterologous expression in Xenopus laevis oocytes, Price et al. (52) identified an amino acid transporter, ApGLNT1, that transports glutamine from hemolymph into bacteriocyte cells. ApGLNT1 is a high-affinity glutamine transporter whose transport of glutamine is inhibited by arginine, an end-product metabolite synthesized by Buchnera. Remarkably, the localization of ApGLNT1 in the bacteriocyte plasma membrane coupled with its transport capabilities strongly suggests ApGLNT1 functions as an important regulator of amino acid biosynthesis in the holobiont. The generality of symbiotic regulation by substrate feedback inhibition in other insect nutritional endosymbiosis requires investigation.

Questions of developmental integration are fundamentally important to advancing understanding of holobiont evolution generally and are particularly important for elucidating the role of constraint in host/symbiont coevolution. Nutritional endosymbiont transmission has been studied best in A. pisum. Like all aphids, A. pisum is cyclically parthenogenetic; thus, a single A. pisum lineage (genotype) has the capacity to reproduce both sexually and asexually during different times of the year. When aphids reproduce sexually, they are oviparous; when they reproduce asexually, they are viviparous. The ease of maintaining a continuous supply of asexual embryos vs. the difficulty of maintaining a steady supply of sexually produced embryos means that most recent work has focused on Buchnera transmission during asexual reproduction (53). That said, during both sexual and asexual reproduction in A. pisum it is clear that Buchnera do not localize to the germarium at any point in development and that each developing oocyte (in the case of sexual reproduction) or embryo (in the case of asexual reproduction) receive Buchnera from a single maternal bacteriocyte cell (13, 53–55). The most recent model of endosymbiont transmission, which was built from examination of transmission electron micrograph images of Buchnera transmission in asexual A. pisum, demonstrates that Buchnera transmission occurs by a process of exo/endocytosis (53). Individual Buchnera are exocytosed from a single maternal bacteriocyte that is closely associated with the posterior end of an embryo at stage 7 of embryogenesis. During exocytosis, Buchnera lose their host-derived symbiosomal membrane and migrate across the “transmission center” as naked Buchnera cells. When they reach the passage between the enlarged follicle cells, they are endocytosed by the multinucleate syncytium within the blastoderm. During endocytosis, the naked Buchnera regain their symbiosomal membrane and move into the central syncytium where they await cellularization before germband formation (55). It is important to note that although Buchnera are closely associated with the germline during development, they never invade the germline. This distinction is significant when considering transmission of the endosymbiont Wolbachia, which infects the germline of its host (56). Although the cell biology of Wolbachia host interactions is particularly well characterized and has benefitted from a lot of careful and elegant work in the model insect Drosophila (e.g., refs. 57–60), we caution at this point against extrapolating mechanisms of Wolbachia transmission to the transmission of bacteriome-restricted nutritional symbionts, because, to the best of our knowledge, bacteriome-associated nutritional symbionts do not infect and are not associated with germline stem cells (13, 55, 61). That said, because in aphid development Buchnera are surrounded by actin filaments (55), the discovery that Wolbachia makes use of the host microtubule network and its associated proteins to move around Drosophila oocytes (57) suggests an exciting line of investigation for those interested in addressing the question of how during development nutritional endosymbionts migrate from bacteriocytes to oocytes (or in the peculiar case of asexual aphids, to embryos).

Two recent studies have investigated the cellular localization of novel bacteriocyte-expressed proteins. In the first, Shigenobu and Stern (62) took a de novo approach to functional characterization of A. pisum orphan proteins, and in doing so they identified novel secreted proteins that are overrepresented in bacteriocyte transcriptomes. The novel secreted proteins fell into two groups that they named bacteriocyte-specific cysteine-rich (BCR) proteins and secreted proteins (SP). Interestingly, all 7 BCR genes and half of the 6 SP genes show identical patterns of in situ hybridization, being first expressed in stage 7 asexual embryos in the syncytium, a location and developmental stage coincident with Buchnera infection of the blastula (55). Expression of all 10 of those genes was maintained in bacteriocyte cells throughout aphid development and in adults. The role these novel secreted proteins play in mediating and maintaining holobiont function is yet to be elucidated. However, this work by Shigenobu and Stern (62) highlights an approach that can usefully be applied to studying developmental integration, an area of investigation that offers both promise and opportunity.

The second study that investigated the cellular localization of novel bacteriocyte-expressed proteins investigated the protein localization of RlpA4 (rare lipoprotein A paralog number 4), an A. pisum gene acquired by lateral gene transfer from an unknown bacterial host (33, 34). Nakabachi et al. (63) found that the protein product of A. pisum genome-encoded RlpA4 is targeted to Buchnera cells in maternal bacteriocytes, a result that has important implications when considering the distinctions traditionally drawn between organelles and endosymbionts. For example, it is commonly argued that an important transition in organelle evolution has been the evolution of mechanisms that facilitate targeting of host genome-encoded proteins back to organelles (64, 65). Thus, the recent discovery that host genome-encoded RlpA4 is targeted to Buchnera cells blurs the distinction between organelles and endosymbionts and further raises the possibility that more host-encoded genes are similarly targeted to endosymbionts in aphids and other holobionts.

In highlighting recent work at the frontier of insect obligate nutritional endosymbiosis research our goal has been to draw attention to advances that have been made in understanding host/symbiont metabolic and developmental integration and also to draw attention to the importance of postgenome approaches to hypothesis testing and exploration. Going forward, the field is especially ripe for the engagement of cell and developmental biologists passionate about understanding the role of symbiosis in evolution.

Conclusion

A new era in symbiosis research arrived in 2010 with publication of the A. pisum genome. Since that time genomics have transformed our understanding of host/endosymbiont relationships. Once understood as mutualisms between biologically autonomous entities, obligate symbiotic relationships are more accurately composed of interdependent parts of an intimately coevolving whole, the holobiont. Despite the current limited availability of paired genomes, these genomes support identification of signatures of holobiont genome evolution. These signatures include host/endosymbiont metabolic collaboration, acquisition of novel genomic material, and evolutionary constraint. As more holobiont genomes are sequenced, we anticipate that these signatures will continue to be supported and that other as yet unidentified signatures will likely emerge. New paired genomes will additionally enhance the shift of symbiosis research toward adoption of functional genomics approaches for enhanced elucidation of the cellular and developmental mechanisms of host/endosymbiont integration. Thus, paired host/symbiont genomes provide the foundation for identification of the fundamental principles of holobiont evolution, advances in knowledge that are shaping our paradigm of what it means to be an organism.